Selective dependency of CALR-mutant myeloproliferative neoplasms on TYK2 signaling

Selective dependency of CALR-mutant myeloproliferative neoplasms on TYK2 signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Selective dependency of CALR-mutant myeloproliferative neoplasms on TYK2 signaling Milena Kalmer, Chiara Wirths, Rebecca Lemanzyk, Alessia Piergentili, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8425696/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Myeloproliferative neoplasms (MPN) are driven by the oncoproteins JAK2V617F, mutant calreticulin (CALR), and mutant thrombopoietin receptor (TPOR), all of which activate JAK/STAT signaling. While JAK2 signaling is engaged in all MPNs, TYK2 is dispensable for JAK2V617F-driven disease. Here, we hypothesized a distinct role for TYK2 in CALR-mutant driven MPN. We found constitutive TYK2 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-expressing cells. Modelling of JAK2/TPOR vs. TYK2/TPOR interaction confirmed similar binding affinities of both kinases to the receptor, which was more relevant in CALRdel52-positive cells. The TYK2 inhibitor deucravacitinib reduced viability and STAT3/5 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-mutant cells, with enhanced efficacy when combined with the JAK2-selective inhibitor fedratinib. Cellular response correlated with JAK2 protein abundance, as CALRins5 JAK2 high clones outcompeted JAK2 low clones upon TYK2 inhibition. In primary samples, deucravacitinib significantly suppressed colony growth in ET and PMF but not PV, and selectively reduced CALR- but not JAK2V617F-mutant allele burden. Similarly, CALR-mutant patient-specific iPSC-derived CD34 + progenitors were more sensitive to TYK2 inhibition than their JAK2V617F counterparts. These findings identify TYK2 as a selective vulnerability in CALR-mutant MPN and support combined TYK2/JAK2 inhibition strategies to overcome JAK2-dependent resistance. Health sciences/Diseases/Haematological diseases/Haematological cancer/Myeloproliferative disease Biological sciences/Stem cells/Haematopoietic stem cells Biological sciences/Cell biology/Cell signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In the classical Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), three prominent driver mutations in the genes of janus kinase (JAK) 2, calreticulin ( CALR ) and the thrombopoietin (TPO) receptor ( MPL gene / TPOR protein) are described 1 , 2 . The JAK2V617F mutation occurs most frequently (95% of PV patients, 50–60% of ET and PMF patients), while CALR frameshift mutations account for 25–35% and MPL mutations for 5–10% of ET and PMF cases, respectively. These driver mutations activate JAKs, explaining the clinical efficacy of the JAK1/2 tyrosine kinase inhibitor (TKI) ruxolitinib 3 – 5 . JAKs associate with various receptors, including the interferon-α receptor (IFNAR). Upon ligand binding to IFNAR, activated TYK2 and JAK1 phosphorylate signal transducer and activator of transcription (STAT) proteins 6 , 7 . While JAK1 is associated with IFNAR2 8 , TYK2 is associated with IFNAR1 9 . Besides its role in IFNα signaling, TYK2 activation is involved in cytokine signaling of the IL-10/IL-20, IL-12 and IL-6/gp130 families 10 – 12 and is implicated in autoinflammatory diseases 13 , where TYK2 inhibitors have shown efficacy and good tolerability 14 – 16 . The TYK2 inhibitor deucravacitinib (BMS-986165) has demonstrated clinical efficacy and a favorable safety profile in phase II/III psoriasis trials (POETYK PSO1) and is approved for moderate-to-severe plaque psoriasis 17 – 20 . Moreover, deucravacitinib decreased pruritus in psoriasis patients by over 70%, a symptom which is also clinically relevant in MPN patients 21 . Yamaji et al established a TYK2 knockout in a JAK2V617F-positive mouse model and demonstrated that the loss of TYK2 did not affect the development of the MPN phenotype but was essential for IFNα efficacy 22 , 23 . However, similar data are missing for CALR-mutant MPN. Here, we analyzed the role of TYK2 in CALR- vs. JAK2V617F-mutant cells and observed increased TYK2 phosphorylation and enhanced susceptibility to TYK2 inhibition of CALR-mutant cells. Thus, our data provide new evidence for selective vulnerabilities of CALR- but not JAK2V617F-mutant cells, suggesting that TYK2 inhibitors may be suited for the treatment of patients with CALR-mutant MPN through improved mutant cell selectivity. Materials and Methods Primary patient samples Peripheral blood samples from MPN patients were obtained from the Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation at RWTH Aachen University Hospital after patients’ written informed consent, as approved by the local ethics committee (EK 127/12). Healthy control samples were obtained from the Department of Transfusion medicine at RWTH Aachen University Hospital after written informed consent (EK099/14). The list of included patient samples and isolation method of peripheral blood mononuclear cells (PBMC) are provided in the Supplementary information (Table S1 ). Colony formation unit (CFU) assay and calculation of clonogenic variant allele frequency (VAF) Execution of CFU assays and calculation of clonogenic VAF is described in detail in the Supplementary information and has been described previously 24 . Cell culture 32D (murine hematopoietic myeloid precursor cell line) and TF-1 (human erythroleukemic cell line) cells (DSMZ, Braunschweig, Germany) transduced with MPL-HA (32D MPL and TF-1 MPL ) and either empty vector (EV), murine JAK2V617F or human type 1 mutant CALRdel52 and type 2 mutant CALRins5 were previously described 25 – 27 , and cultured in RPMI1640 medium containing 10% FCS, 10% WEHI supernatant as source of IL-3 (32D) or 2 ng/ml GM-CSF (TF-1) and 1% Pen/Strep. Prior to experiments, cells were IL-3- or GM-CSF-starved, respectively, for 24 h. Antibodies and Reagents Deucravacitinib (BMS-986165; MedChemExpress, USA) and ruxolitinib (LC Laboratories, USA) were dissolved in DMSO. Murine IFNα, dissolved in ddH2O, was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies used for Western Blotting are listed in table S2 . CRISPR/Cas9 targeting of Tyk2 Two strategies were applied to target the Tyk2 gene in 32D cells, as described in Fig. 2 A and the Supplementary information (Fig. S1 -2). Potential off-targets of the CRISPR/Cas9 procedure were excluded by Sanger sequencing (Fig. S3). Cell lysates, SDS-Page and Western Blotting Preparation of whole cell lysates, SDS-Page and Western Blot (WB) were performed as previously described 28 . ImageJ or GelAnalyzer (ver. 23.1.1) software was used for quantification of protein levels. MTT assay After washing cells twice with PBS, 3 x 10 4 cells/well were plated in triplicate on 96 well plates. IFNα and deucravacitinib or fedratinib were diluted in cell culture medium to concentrations ranging from 1–10,000 U/ml and 0.01 to 10 µM, respectively. DMSO served as control. Metabolic activity of the cells was measured using MTT reagent (Sigma-Aldrich) after 72 h of incubation at 37°C at an absorption of 550 nm. Apoptosis staining 2 x 10 5 cells per ml were treated with 5 µM deucravacitinib for a total duration of 48 h. Apoptosis staining was performed with Annexin V-APC and 7-AAD (1:100 dilution; Biolegend). Samples were analyzed with a Gallios flow cytometer (Beckmann Coulter, Krefeld, Germany). Differentiation of induced pluripotent stem cells (iPSC) into hematopoietic stem and progenitor cells (HSPC) and Cell Viability Assay iPSC-derived MPN clones (JAK2V617F het09, CALRdel52 het01 and CALRins5 het04) were differentiated into HSPC using the spin-EB method as described previously 29 , 30 . On day 14, CD34 + HSPCs were enriched by magnetic activated cell sorting (MACS) according to manufacturer’s protocol (Miltenyi, Bergisch-Gladbach, Germany). The Cell Viability Assay is described in the Supplementary information. Statistical analysis All experiments were performed at least three times unless stated otherwise. Statistical differences were calculated using the tests as indicated in the figure legends, with p < 0.05 considered significant. Results TYK2 is constitutively phosphorylated in CALR- but not in JAK2V617F-mutated cells Previously, our group investigated differences between CALR- and JAK2V617F- mutant cells in their sensitivity to IFNα treatment illustrating differential JAK1 activation 26 , 31 . Given the limited data on the role of TYK2, particularly in CALR-mutated MPN, and the close interaction of both TYK2 and CALR with TPOR, we compared the basal activity of TYK2 in JAK2V617F-mutant versus CALRdel52 cells. 32D MPL EV, JAK2V617F, or CALRdel52 cells were analyzed for basal phosphorylation of TYK2 (pTYK2). We found pTYK2 in 32 MPL CALRdel52 cells (Fig. 1 A), which was significantly higher in comparison to 32 MPL JAK2V617F cells. Increased pTYK2 was confirmed in human TF-1 MPL CALRdel52 in comparison to JAK2V617F cells (Fig. 1 B). Mutated CALR selectively activates signaling via the TPOR, while the JAK2V617F oncogene interacts with a broader array of receptors 32 , implying a direct involvement of TPOR in TYK2 activation downstream of mutated CALR. A model based on the structure of the TYK2 FERM-SH2 domain (PDB ID: 4PO6) in complex with TPOR (red) as well as JAK2 FERM-SH2 domain (PDB ID: 6E2Q) was generated and simulated by molecular dynamics (MD) (Fig. 1 C and see Supplementary Methods). Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations were performed on the MD simulations to estimate binding free energies. Our calculations indicate that TYK2 binds TPOR with comparable affinity to JAK2 (ΔG ≈ − 205 kcal/mol vs − 209 kcal/mol), supporting that TYK2 contributes functionally to TPOR signaling, alongside JAK2 (Table 1). We therefore examined whether TPO stimulation activates TYK2, to determine if the elevated basal pTYK2 levels are associated with constitutive TPOR signaling. TPO stimulation induced pTYK2 in 32D MPL EV, JAK2V617F and CALRdel52 cells (Fig. 1 D), but the extent of induction was least pronounced in CALRdel52 cells. In addition, upon TPO stimulation, pJAK2 and downstream pSTAT3/5 increased in EV and JAK2V617F but not CALRdel52 cells, while basal STAT phosphorylation was similar between JAK2V617F and CALRdel52. JAK2 whole protein expression was higher in JAK2V617F cells due to the ectopic expression of the oncogene. Interestingly, TPO stimulation strongly activated downstream signaling, including pTYK2, in healthy donor platelets, whereas responses in MPN platelets were weaker and highly variable (Fig. S4A). This likely reflects the significantly reduced TPOR and CD41/CD61 surface expression on ET and MF platelets, independent of driver mutation (Fig. S4B). Together, these results demonstrate a direct link between TPOR signaling and TYK2 phosphorylation and highlight the potential relevance of pTYK2 in CALRdel52-mutated cells. Furthermore, TPO stimulation mimics basal CALRdel52-activation of TYK2 downstream of TPOR, which can be amplified in JAK2V617F-expressing cells. TPOR/TYK2 interaction is crucial for CALRdel52-induced signaling To further investigate the necessity of TYK2 activation and its TPOR interaction in 32D MPL JAK2V617F and CALRdel52 cells, two knockout strategies using CRISPR/Cas9 were applied, both aiming at the FERM domain, which facilitates the interaction of TYK2 with its respective receptors (Fig. 2 A) 33 . Sanger sequencing of the PCR-amplified TYK2 cDNA revealed that CRISPR strategy 1 yielded clones with small and large deletions (Fig. S1 A-D). In JAK2V617F- and CALRdel52-mutated background, clones were generated lacking TYK2 phosphorylation. Nevertheless, total TYK2 protein could still be observed in all clones. In a second attempt, aiming to completely eliminate TYK2 protein, we applied CRISPR strategy 2 (Fig. 2 A). We again recovered pTYK2-deficient clones, however, these were confined to JAK2V617F- and not CALR-mutated 32D MPL cells (Fig. S2 A-E). Therefore, we hypothesized that the induced genetic changes via strategy 1 were less severe on downstream signaling compared to strategy 2, explaining the absence of strategy 2-derived 32D MPL CALRdel52 cell clones. Since TYK2 is a key mediator of IFNAR1/2 signaling and IFNα inhibits 32D cell growth, we assessed the IFNα response of strategy 1 and strategy 2 mutants using an MTT assay. As expected, JAK2V617F strategy 1 (p < 0.05) and strategy 2 (p < 0.0001) cells were significantly less sensitive than their parental counterparts (Fig. 2 B). In line with our hypothesis, pTYK2-deficient JAK2V617F clones resulting from strategy 2 showed almost no reduction of viability in response to IFNα and this reduction in response was significantly more pronounced than in strategy 1-derived clones (IFNα 1000 U; p < 0.05). 32D MPL CALRdel52 cells were not informative here and remained IFNα-insensitive, as described before 26 . To confirm the relevance of pTYK2 for TPO downstream signaling in CALR-mutated cells, we stimulated 32D MPL JAK2V617F or CALRdel52 parental cells and strategy 1 and strategy 2 clones with TPO. Apart from complete loss of pTYK2, phosphorylation of STAT factors and JAK2 was preserved in strategy 1 JAK2V617F but not CALRdel52 cells, highlighting the importance of TYK2 for TPOR/CALRdel52 signaling (Fig. 2 C). JAK2V617F strategy 2 cells showed comparable basal downstream signaling to parental JAK2V617F cells, but were less inducible by TPO, supporting our hypothesis of these being more impaired in their downstream signaling (Fig. 2 C). Next, the strategy 2 deletion (Fig. S2 A; aa del158-223), observed in 32D MPL JAK2V617F cells was modeled on the TYK2-TPOR complex structure, and MD simulations were performed. Increased structural instability of TYK2 mutant del158-223 complex versus TYK2 WT was reflected by fewer hydrogen bonds throughout the simulation at the TYK2-TPOR interface (Fig. 2 D). The deletion of aa 158–223 induced changes in the aa sequence of the F2 domain and conformational changes in the F1 domain and the linker L1, all part of the FERM domain facilitating the receptor interaction 33 . MM-GBSA analysis further supported reduced TPOR binding of the TYK2 del158–223 mutant, which showed a markedly lower predicted affinity than WT TYK2 (ΔG − 154.8 ± 31.3 vs. − 205.2 ± 22.4 kcal/mol; Table 1, Fig. 2 D). These data indicate that del158–223 destabilizes TPOR–TYK2 interaction, likely explaining the inability to generate CALRdel52 32DMPL clones using CRISPR strategy 2. Combination of TYK2- and JAK2-selective inhibitors show synergistic effects Given that TYK2 was selectively activated in CALR-mutant but not JAK2V617F-mutant 32D MPL cells (Fig. 1 A), we analyzed the efficacy of deucravacitinib, a specific TYK2 inhibitor, including 32D MPLW515K cells, hypothesizing that they may respond similarly to CALRdel52 cells upon TYK2 inhibition. We confirmed a significant drop of viability of CALRdel52 and MPLW515K but not JAK2V617F cells (Fig. 3 A). Correspondingly, we observed a significant increase of apoptosis of 32D MPL CALRdel52 but not JAK2V617F cells upon deucravacitinib treatment (Fig. 3 B). Comparable constitutive pTYK2 levels in CALRdel52 and MPLW515K cells were confirmed, which were abolished by deucravacitinib treatment (Fig. 3 C). In line with these results, pSTAT1, pSTAT3 and pSTAT5 levels were downregulated only in CALRdel52 and MPLW515K but not JAK2V617F cells (Fig. 3 C). Ruxolitinib’s efficacy partly relies on its anti-inflammatory JAK1/JAK2 inhibition 34 , whereas the more JAK2-selective inhibitors fedratinib and pacritinib may be promising partners for deucravacitinib. We therefore treated 32D MPL JAK2V617F and CALRdel52 cells with 0.5 µM deucravacitinib plus increasing doses of fedratinib for 72 h and assessed viability (Fig. 3 D). The combination synergistically reduced viability in CALRdel52 but not JAK2V617F cells (coefficient of drug interaction (CDI) 35 below 1 considered as synergistic; 0.68 for 0.5 µM + 0.5 µM; 0.38 for 0.5 µM and 1 µM). Downstream signaling was assessed following treatment with 1 µM deucravacitinib, 0.5 µM fedratinib or their combination (Fig. 3 E). In JAK2V617F expressing cells, phosphorylation of STAT proteins was not altered upon these low inhibitor concentrations (Fig. 3 E). Like ruxolitinib, fedratinib is a type I TKI leading to a paradoxical hyperphosphorylation of the regulatory tyrosines in JAK2 upon treatment 36 , 37 . 32D MPL CALRdel52 and MPLW515K cells were most sensitive to deucravacitinib and the combination treatment as demonstrated by a strong downregulation of pSTAT3 and pSTAT5 (Fig. 3 E; Fig. S5). Expression of CALRins5 led to phosphorylation of pTYK2, and the signal was ablated upon deucravacitinib treatment, while pSTAT3 and pSTAT5 were less targeted by the combination treatment. Endogenous JAK2 levels define biologic deucravacitinib efficacy Strikingly, these 32D MPL CALRins5 cells expressed higher levels of endogenous JAK2 in comparison to CALRdel52 and MPLW515K expressing cells (Fig. 3 E). The high levels of endogenous JAK2 protein in CALRins5-expressing cells may be a factor that affect responsiveness to deucravacitinib, given that TYK2 and JAK2 may compete for TPOR binding 38 , 39 . Therefore, we compared the efficacy of deucravacitinib in CALRdel52 vs CALRins5 cells and demonstrated significant differences in sensitivity (Fig. 4 A). Deucravacitinib efficiently reduced pTYK2 and downstream signaling in CALRdel52 cells but was less efficient in 32D MPL CALRins5 and JAK2V617F cells (Fig. 4 B). STAT5 phosphorylation, in particular, remained unaffected, and again overall JAK2 protein levels were higher in CALRins5 cells in comparison to CALRdel52 cells. To confirm the importance of JAK2 protein expression in the differential response to deucravacitinib, we ectopically overexpressed murine JAK2 WT in 32D MPL CALRdel52 cells 26 and demonstrated no response to the TYK2 inhibitor up to a concentration of 10 µM (Fig. S6A). Next, we performed single cell dilutions of the 32D MPL cell line to discriminate clones with high and low JAK2 protein expression (Fig. S6B). Three cell clones each with low or high JAK2 protein levels were stably transduced with CALRins5. Two clones each were treated with deucravacitinib, and clones #2 and #6 were confirmed to harbor low, and clones #3 and #8 high JAK2 protein levels (Fig. 4 C). In line with our hypothesis, clones #2 and #6 showed reduced pSTAT3, pSTAT5 and pTYK2 levels and strong sensitivity to deucravacitinib treatment (Fig. 4 C; Fig. S6C). In contrast, clones #3 and #8 showed an inverse response. To analyze clonal advantage during deucravacitinib treatment, mCherry expressing 32D MPL CALRins5 #3 cells were generated (high JAK2), mixed 1:1 with 32D MPL CALRins5 #6 cells (low JAK2) and treated with deucravacitinib for 48 h. Clonal growth was analyzed by discriminating between GFP+ (#6) and GFP+/mCherry+ (#3) cells in flow cytometry. Although 32D MPL CALRins5 #3 cells started out with a slightly lower percentage, #3 cells outcompeted #6 cells in the presence of the TYK2 inhibitor (Fig. 4 D), demonstrating that high JAK2 expression may overcome the sensitivity of CALR-mutant cells to TYK2 inhibition. The JAK2, CALRins5, TPOR clone resists deucravacitinib treatment When assessing 32D MPL CALRins5 clones by flow cytometry for GFP and TPOR surface expression, two populations with differential ratios were observed: for #3 and #8, the main population was GFP med (corresponds to CALRins5 med ) and TPOR high . In comparison, the main population in clones #2 and #6 was GFP high (CALRins5 high ) and TPOR med (Fig. S7). We hypothesized that the GFP med (CALRins5 med ) and TPOR high population is also JAK2 high , correlating with the JAK2 protein levels in CALRins5 #3 and #6 cells confirmed by WB. As both populations are present in both clones to different extents, we treated 32D MPL CALRins5 #3 and #6 with deucravacitinib for 48 h and 96 h and analyzed GFP/TPOR positivity (Fig. 4 E, left). Strikingly, in CALRins5 #6, the GFP high /TPOR med (expected to be JAK2 low ) significantly decreased and in CALRins5 #3, the GFP med /TPOR high (expected to be JAK2 high ) population increased (Fig. 4 E, right). Hence, the combination of lower CALRins5, high TPOR surface expression and high JAK2 levels showed resistance to TYK2 inhibition and reduced TYK2-dependency. Phenotypic response to deucravacitinib is driven by the dependence on TPOR but not EPOR Next, we investigated whether the CALR-specific effects observed in 32D MPL cells also translate to primary patient-derived cells. Therefore, isolated PBMC from MPN patients and healthy controls (HC) were seeded in colony-formation assays with and without deucravacitinib (Table S1 ). Colonies were counted 10–13 d after seeding, and the number of colonies in drug condition was normalized to the untreated condition. Overall, colony growth was significantly reduced upon treatment (Fig. S8A). Grouping by diagnosis revealed a stronger deucravacitinib effect in ET compared with HC and PV, and in PMF compared with PV, with a trend toward greater sensitivity than HC (p = 0.0954) (Fig. 5 A). As PV is driven mainly by erythrocytosis and enhanced EPO signaling, whereas ET and PMF are linked to aberrant megakaryopoiesis and constitutive TPO signaling, we next tested TYK2 inhibition in 32D cells expressing either EPOR or TPOR. Consistent with patient data, deucravacitinib reduced metabolic activity significantly less in EPO-dependent than in TPO-dependent cells (Fig. 5 B). Next, we examined whether the response to deucravacitinib was genotype-dependent and found that colony growth was comparable across driver oncogene genotypes (Supplementary Fig.S8B). However, when analyzing the fraction of mutated colonies, higher sensitivity of CALR-mutant in comparison to JAK2V617F-mutant colonies was confirmed, as demonstrated by a significantly more pronounced reduction of calculated CALR- vs. JAK2-mutant clonogenic VAF (Fig. 5 C; Fig. S8C). To confirm genotype-related effects of TYK2 inhibition, JAK2V617F-, CALRdel52- and CALRins5-mutant induced pluripotent stem cells (iPSC), that we had previously generated from MPN patients 29 , 30 , were analyzed. To do this, iPSC-derived CD34 + HSPC were treated with deucravacitinib (DMSO, 0.5, 1 and 5 µM) for 72 h, and assessed for cell viability. While CALR-mutated cells were sensitive to TYK2 inhibition, showing significantly reduced viability, JAK2V617F-expressing iPSC-derived CD34 + HSPC were resistant to deucravacitinib treatment (Fig. 5 D). Overall, these data indicate that CALRdel52-mutated cells are sensitive to TYK2 inhibition, and underscore the influence of disease phenotype and the differential involvement of EPOR versus TPOR signaling. Discussion Until now, TYK2 is primarily known for its role in different autoimmune and inflammatory diseases 40 , 41 . Here, we suggest TYK2 to be a novel pharmaceutical target in CALR-mutant MPN. The JAK1/JAK2 inhibitor ruxolitinib is approved for the treatment of myelofibrosis and hydroxyurea-resistant or -intolerant PV, and many patients show excellent hematologic responses including reduction of elevated blood counts, splenomegaly and overall symptom burden 42 . Next to JAK2 and JAK1, ruxolitinib also inhibits TYK2 activity in vitro , with an IC50 of 19 nM 23,43,44 . As we observed constitutive TYK2 phosphorylation selectively in CALR- vs. JAK2V617F-mutant cells, the efficacy of ruxolitinib on CALR-mutated MPN may at least partly be attributed to TYK2 inhibition. When stimulating TPOR-expressing cell lines, patient- and HD-derived platelets with TPO, TYK2 was strongly phosphorylated, demonstrating the direct activation of TYK2 downstream of TPOR. Interestingly, our structural model and energy analysis of TPOR/TYK2 and TPOR/JAK2 showed a similar ΔG, which suggests that JAK2 and TYK2 bind the receptor with comparable affinity. TPO-induced TYK2 phosphorylation was low in CALRdel52-expressing cells, likely because mutant CALR binds the TPOR extracellular domain, mimicking cytokine engagement and limiting additional TPO-driven signaling 45 , 46 . These findings suggest that CALRdel52 activates TYK2 by inducing a TPOR intracellular conformation similar to that triggered by TPO binding. This would be in line with Papadopoulos et al, suggesting that mutant CALR effectively replaces TPO as an artificial ligand 47 . Nevertheless, the suggested tetrameric mutant CALR-TPOR architecture contrasts with 1:2 TPO:TPOR cryo-EM structures, described by Tsutsumi et al, highlighting potential structural differences at the intracellular JAK binding sites 48 . In contrast, JAK2V617F binding to TPOR may promote a distinct C-terminal conformation that favors JAK2/receptor association. We aimed to generate TYK2-deficient JAK2V617F- and CALRdel52-mutant 32D MPL cells utilizing two different CRISPR/Cas9 knockout strategies (nickase or nuclease). With both strategies, pTYK2-deficient 32D MPL JAK2V617F cell lines were successfully established, but in 32D MPL CALRdel52 cells, only strategy 1 was successful. No complete TYK2 knockout was obtained in any of the cell lines. Strategy 2 yielded clones that were most strongly impaired in their downstream signaling when treated with TPO or IFNa, and when analyzed by MD simulations. Deucravacitinib, a selective TYK2 inhibitor, targets the pseudokinase domain of TYK2, which makes it more specific than ruxolitinib 49 . Inhibition of TYK2 had a significantly stronger effect on CALRdel52- and MPLW515K-expressing cells compared to JAK2V617F-mutant cells, demonstrating the relevance of TYK2 for TPOR-driven signaling in these cells. Oncogenic signaling of CALR mutations relies on TPOR 50 , 51 , whereas JAK2V617F also interacts with EPOR and CSF3R 52 . TYK2 has been reported to associate with TPOR and, like JAK2, regulate its surface expression 39 , 53 , 54 . Reduced TYK2 activity or binding affinity could thus lower TPOR surface levels. Likewise, TYK2 deficiency reduces IFNAR1 surface expression by driving it into perinuclear compartments 55 . Nevertheless, TYK2 inhibition by deucravacitinib did not alter TPOR surface expression in JAK2V617F- or CALR-mutant 32D MPL cells (data not shown). To our surprise, deucravacitinib did not reduce cell viability of 32D MPL CALRins5 expressing cells, and it showed less effect on downstream signaling in these cells. The reduced response to TYK2 inhibition was attributable to higher endogenous JAK2 protein levels in 32D MPL CALRins5 clones compared with CALRdel52 clones. We confirmed that the 32D MPL CALRins5 JAK2 high clones were less sensitive to TYK2 inhibition and outcompeted JAK2 low cells in co-culture with deucravacitinib. In addition, a dominant surface TPOR high /GFP med population was present in deucravacitinib-resistent clones, while a TPOR med /GFP high population was dominant in deucravacitinib-sensitive clones. All clones showed a similar basal pTYK2 band in WB. Hence, pTYK2 was not the main driver of TPOR surface expression but rather JAK2 and the amount of CALRins5 expression (indicated by GFP). Chachoua and colleagues described reduced maturation of TPOR in mutant CALR expressing cells 56 , thereby reducing surface trafficking, which may then depend on the amount of mutant CALR per cell. Overall, in patients with CALR mutations, particularly with TPOR med JAK2 WT low CALRmut high , the HSPC population may especially benefit from deucravacitinib treatment. However, the selection of JAK2 high clones under deucravacitinib treatment highlights the importance to target simultaneously JAK2 and TYK2. Combination of deucravacitinib and fedratinib induced synergistic effects in 32D MPL CALRdel52 cells. Although combination of different JAK inhibitors has been discussed, particularly in MPN and autoimmune diseases, studies are currently still preclinical or entering early clinical testing 57 , 58 . Special caution would be required due to overlapping immunosuppression and a potential increased risk of infection or cytopenia. Experiments on primary MPN patient material confirmed selective sensitivity to TYK2 inhibition in CALR-mutant cells from patients with ET and PMF, but not those from patients with JAK2V617F-mutant cells and those with PV harboring a JAK2 mutation. Mechanistically, we linked the selective TYK2 dependence to those MPN, where the TPOR (and not EPOR) is the main driver of cell proliferation. Importantly, we showed that TYK2 inhibition led to a significantly stronger decrease in the calculated VAF in CALR- vs. JAK2V617F-mutant clonogenic cells and even more immature CD34 + iPSC-derived HSPC. In summary, our data provides the rationale for a new therapeutic approach for patients with MPN, particularly those harboring CALR mutations. Deucravacitinib is approved for the treatment of patients with psoriasis, demonstrating good clinical activity and acceptable toxicity 14 , with no relevant anemia or thrombocytopenia. As TYK2 plays an important role in the persistence of JAK2V617F-positive cells during ruxolitinib therapy 59 , it might be interesting to analyze the effects of a double treatment with JAK2- and TYK2-specific inhibitors. In MPN patients suffering from (ro)pegIFNα-induced pruritus, deucravacitinib, which has been shown to reduce itch in psoriasis patients by over 70% in two weeks, could be used to treat this pruritus 21 . In those MPN cases of concomitant TKI and IFNα therapy, TYK2 inhibition may partly counteract the IFNα effects. However, this was not seen in the clinical setting for ruxolitinib and pegylated IFNα 60 , possibly due to differential kinetics of pegIFNα and the JAK inhibitor 21 . Finally, deucravacitinib may be an ideal combination partner for the newly-developed mutant-CALR-specific antibodies, which are currently in clinical trials (NCT05936359 and NCT06150157). Declarations Acknowledgements This work was in part funded by research grants to SK from the Deutsche Forschungsgemeinschaft (German Research Foundation) (DFG KO 2155/6-1, project number AOBJ 636363) (DFG KO 2155/8-2, project number AOBJ: 680695) and by funds from the German Research Foundation as part of the Clinical Research Unit CRU 344 to S.K. (KO2155/7-1, project number 428858786) and N.C. (CH1509/1-1) as well as the Deutsche José Carreras Leukämie-Stiftung (DJCLS R 16/2017). This work was supported by RWTH Aachen University through Seed Funds Project OPSF702 of G.R. and N.C.. The work was partly performed in the Core Facility Flow Cytometry, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University. Biomaterial samples were provided by the RWTH centralized Biomaterial Bank Aachen (RWTH cBMB, Aachen, Germany) in accordance with the regulations of the biomaterial bank and the approval of the ethics committee of the medical faculty, RWTH Aachen. G.R. and A.P. acknowledge the Federal Ministry of Education and Research (BMBF) and the state of North Rhine-Westphalia as part of the NHR Program. Part of this work was generated within the medical thesis works of RL, LS, and CW. MK is supported by a postdoctoral fellowship from de Duve Institute and by a Fonds Speciaux de recherches fellowship of UCLoouvain. Funding to SNC is acknowledged from Ludwig Institute for Cancer Research, Fondation contre le cancer, Salus Sanguinis and Fondation “Les avions de Sébastien”, projets Action de recherché concertée (ARC) 16/21-073, Projet de recherche FNRS n°T.0043.21 and WelBio F 44/8/5 - MCF/UIG – 10955. Conflict of interest Steffen Koschmieder received research funding from Geron, Janssen, AOP Pharma, and Novartis; received consulting fees from Pfizer, Incyte, Ariad, Novartis, AOP Pharma, Bristol Myers Squibb, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, GSK, Sierra Oncology, and PharmaEssentia; received payment or honoraria from Novartis, BMS/Celgene, Pfizer, AstraZeneca, and iOMEDICO; received travel/accommodation support from Alexion, Novartis, Bristol Myers Squibb, Incyte, AOP Pharma, CTI BioPharma, Pfizer, Celgene, Janssen, Geron, Roche, AbbVie, GSK, Sierra Oncology, Kartos, AstraZeneca, Protagonist, and iOMEDICO; had a patent issued for a BET inhibitor at RWTH Aachen University; participated on advisory boards for Pfizer, Incyte, Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, GSK, Sierra Oncology, and PharmaEssentia. 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Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling. Leukemia 2019. doi: 10.1038/s41375-019-0427-7 . Koppikar P, Bhagwat N, Kilpivaara O, Manshouri T, Adli M, Hricik T et al. Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy. Nature 2012; 489: 155–9. Sørensen AL, Mikkelsen SU, Knudsen TA, Bjørn ME, Andersen CL, Bjerrum OW et al. Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study. Haematologica 2020; 105: 2262–2272. Table Table 1. MM-GBSA calculations of the predicted binding free energy (ΔG) along the simulations of TPOR in complex with JAK2 WT, TYK2 WT, TYK2 del158-223. Complex with TPOR Δ G in kcal/mol (mean ± SD) JAK2 WT -209.75 ± 28.74 TYK2 WT -205.17 ± 22.43 TYK2 del158-223 -154.76 ± 31.34 Additional Declarations Yes there is potential conflict of interest. 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1","display":"","copyAsset":false,"role":"figure","size":3973303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated TYK2 phosphorylation in CALRdel52-mutant cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Lysates of untreated 32D\u003csup\u003eMPL\u003c/sup\u003e empty vector (EV), JAK2V617F- and CALRdel52-mutant cells were generated and subjected to SDS-PAGE followed by immunoblotting (left). GAPDH served as loading control. Densitometric analyses of three independent WB to confirm the upregulation of pTYK2 relative to TYK2 in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52-mutant cells (right). Statistical analysis was performed using One-way-ANOVA followed by Dunnett's multiple comparisons test. \u003cstrong\u003eB\u003c/strong\u003e Untreated TF-1\u003csup\u003eMPL\u003c/sup\u003e empty vector (EV), JAK2V617F- and CALRdel52-mutant cells were immunoblotted (left) and densitometric analyses of three independent WB (right) was generated. Statistical analysis was performed using One-way-ANOVA followed by Dunnett's multiple comparisons test. \u003cstrong\u003eC\u003c/strong\u003e Model of TPOR in complex with TYK2 FERM-SH2 domains (left) and with JAK2 FERM-SH2 domains (right). Domains are indicated.\u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e EV, JAK2V617F or CALRdel52 cells were treated for 1h with 20\u0026nbsp;ng/ml human TPO. 32D\u003csup\u003eMPL\u003c/sup\u003e EV were WEHI-starved 24h before stimulation. Cells were then harvested, lysates were prepared and analyzed by WB. GAPDH served as loading control. ns = not significant, *p\u0026lt;0.05; ** p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1small.png","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/58d2177e2640b41ab2beb8e5.png"},{"id":99795398,"identity":"17170400-46e1-45d0-a60a-74380a2d12de","added_by":"auto","created_at":"2026-01-08 13:37:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2918500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9-generated TYK2 mutants illustrate relevance of TYK2 for CALRdel52-signaling. A\u003c/strong\u003e Utilized CRISPR/Cas9 strategies are schematically shown. On protein level TYK2-receptor interaction is facilitated by FERM and SH2 domains. The FERM domain is encoded by exon 3 through 9. In CRISPR strategy 1 two guides, binding in exon 4 and 5 and a Cas9 nuclease were used. For CRISPR strategy 2, four guides binding in exon 6 and the adjacent intron and Cas9 nickase were used. \u003cstrong\u003eB\u003c/strong\u003e Parental bulk as well as strategy 1 and 2-derived clones were treated with increasing doses of IFNα (1 to 10000 U/ml IFNα) for 72 h. Metabolic activity of the clones was measured using MTT assay. Statistical analysis was performed using One-way-ANOVA followed by Dunnett’s multiple comparisons test and significances each refer to the parental bulk within the same. \u003cstrong\u003eC\u003c/strong\u003e Analysis of TPO downstream signaling in parental vs. strategy 1 and 2 clones. Cells were stimulated with 10 ng/ml TPO for 30 min, harvested and applied to immunoblotting. \u003cstrong\u003eD \u003c/strong\u003eNumber of hydrogen bonds between TYK2 and TPOR over 500ns MD simulations for WT (green) and del158-223 (orange) systems. Solid lines indicate the smoothed running average, shaded areas indicate fluctuations over time; density distribution of hydrogen bonds over time (right). n=3. *p\u0026lt;0.05; ** p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2small.png","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/e5861b72b31ee0159285753f.png"},{"id":99680317,"identity":"a6cf5082-be36-4ba1-8587-202a68e60656","added_by":"auto","created_at":"2026-01-07 08:51:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3305056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTYK2 inhibition targets TPOR-dependent oncogene-expressing cells but not JAK2V617F-mutated cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F-, CALRdel52- and 32D MPLW515K mutant cells were treated with increasing doses deucravacitinib (deuc) ranging from 0.01 to 10 µM. After 72 h, metabolic activity of the cells was measured using MTT assay. Statistical analysis was performed using unpaired t-tests. n=3. \u003cstrong\u003eB\u003c/strong\u003e Induction of apoptosis upon deuc treatment (5\u0026nbsp;µM, 48\u0026nbsp;h) was analyzed using AnnexinV/7AAD staining via flow cytometry. Statistical analysis was done using unpaired t-test. n=3 \u003cstrong\u003eC\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F-, CALRdel52- and 32D MPLW515K-mutant cells were treated with 0.5 µM deucravacitinib (deuc) for 4 h. After harvesting the cells and preparation of lysates, SDS-PAGE was performed followed by immunoblotting. GAPDH served as loading control. \u003cstrong\u003eD\u003c/strong\u003e 32D\u003csup\u003eMPL \u003c/sup\u003eJAK2V617F or CALRdel52 cells were seeded in medium with or without 0.5 µM deuc and increasing concentrations of fedratinib (Fed). Metabolic activity was assessed after 72 h using MTT. Statistical analysis was performed using One-Way ANOVA. n=3 \u003cstrong\u003eE\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e cells carrying JAK2V617F, CALRdel52, CALRins5 or 32D MPLW515K were treated with 1\u0026nbsp;µM deuc or 0.5\u0026nbsp;µM fedratinib or a combination of both for 4\u0026nbsp;h\u003cstrong\u003e, \u003c/strong\u003ecells were harvested and their lysates were applied to immunoblotting. Downstream signaling effect was analyzed and GAPDH used as a loading control.*p\u0026lt;0.05; **p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3small.png","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/540abb1aed27b46ad3918d28.png"},{"id":99680326,"identity":"363e4f10-792a-42d3-b7d8-021ffc83bb84","added_by":"auto","created_at":"2026-01-07 08:51:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3687865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSensitivity to TYK2 inhibition is determined by JAK2 expression.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 and CALRins5 cells were seeded with increasing concentrations of deuc. Viability was assessed after 72 h using MTT. Statistical analysis was done using an unpaired t-test. n=3 \u003cstrong\u003eB\u003c/strong\u003e Downstream response to deuc treatment was assessed in 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F, CALRdel52 and CALRins5 cells. Cells were treated with 1 µM deuc for 4\u0026nbsp;h, and lysates were analyzed via immunoblotting. GAPDH served as a loading control. n=3 \u003cstrong\u003eC\u003c/strong\u003e 32D\u003csup\u003eMPL\u003c/sup\u003e clones expressing low (#2 and #6) or high (#3 and #8) levels of JAK2 were stably transduced with CALRins5. Cells were treated with 5\u0026nbsp;µM deuc for 4\u0026nbsp;h and lysates were applied to immunoblotting. n=3 \u003cstrong\u003eD\u003c/strong\u003e CALRins5 #3 with JAK2 high expression was stably transduced with mCherry. #3 and #6 were mixed in a 1:1 ratio and cultivated over 48\u0026nbsp;h in the presence of 1\u0026nbsp;µM deuc. Relative ratio of cells was analyzed using flow cytometry in triplicates determining GFP-single vs mCherry-GFP-double positive fraction. Statistical analysis was performed using unpaired t-test at 0\u0026nbsp;h and 48\u0026nbsp;h. n=3 \u003cstrong\u003eE\u003c/strong\u003e GFP and TPOR surface levels were assessed in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 JAK2 high (#3) and JAK2 low (#6) cells via flow cytometry. Cells were cultivated in the presence of 1\u0026nbsp;µM deuc for up to 96\u0026nbsp;h and development of GFP and TPOR surface levels were followed (left). Portion of the different populations were compared between untreated and 48\u0026nbsp;h or 72\u0026nbsp;h deuc treatment and fold change was calculated (right). Statistical analysis was performed using Two-Way ANOVA. n=3. *p\u0026lt;0.05; **p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4small.png","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/3bb2694dcb133c46f3337e64.png"},{"id":99795248,"identity":"40d54940-f0f2-40f8-8773-77331eba93aa","added_by":"auto","created_at":"2026-01-08 13:37:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1106391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTYK2 inhibition confers a stronger effect on CALR-mutant than on JAK2V617F-mutant patient and iPSC-derived CD34+ cells.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003ePBMCs were isolated from patient samples and 1x10\u003csup\u003e6\u003c/sup\u003e cells/ml for ET, PV and HC and 250.000 cells/ml for PMF patients, respectively, were seeded in methylcellulose and treated for 10 to 13 days with 1 µM deuc or DMSO (control). Colonies were counted and DMSO control was compared to treated cells. Relative number of colonies after deuc treatment are divided into MPN subtypes and are shown using a Box-whisker-Plot. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. HC n=3; PV n=4; ET n=9; PMF n=5. \u003cstrong\u003eB \u003c/strong\u003e32D transduced with either MPL or EPOR were seeded in presence of the corresponding cytokine and increasing concentrations of deuc. After 72 h metabolic activity was measured via MTT. Statistical analysis was performed using unpaired t-tests. n=3. \u003cstrong\u003eC\u003c/strong\u003e 30 single colonies from each CFU assays with ET and PMF patient material were picked either DMSO or deuc treated and genotyped for their specific driver mutation by PCR. Boxplot shows change in clonogenic variant allele frequency (VAF) for JAK2V617F and CALRdel52-mutant patients after treatment with deuc. Statistical analysis was performed using Mann-Whitney test. \u003cstrong\u003eD\u003c/strong\u003e iPSC-derived CD34+ cells carrying the JAK2V617F, CALRdel52 or CALRins5 mutation were cultivated in increasing concentration of deuc for 72 h or DMSO. Cell viability was analyzed using CellTiterGlo assay. Statistical analysis was done using Two-way ANOVA. *p\u0026lt;0.05; **p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5small.png","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/460020efa20a44e8a0291432.png"},{"id":107103020,"identity":"76737d6c-95f9-414b-a5aa-a87a3eef5826","added_by":"auto","created_at":"2026-04-16 19:46:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15170066,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/9b55e2fb-589e-4042-9145-504f5f83b4cc.pdf"},{"id":99795708,"identity":"d09a3c08-8a34-4836-9795-c71934418a6b","added_by":"auto","created_at":"2026-01-08 13:39:26","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1514977,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFiguresKalmeretal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/88e7d77bccb1d7f57052ca14.pdf"},{"id":99680310,"identity":"d7ee9618-386f-43cf-920c-c9ff7f5981f5","added_by":"auto","created_at":"2026-01-07 08:51:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43957,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"20251220SupplementaryinformationKalmeretal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8425696/v1/8f4864fdcbc8da4a91dca3cf.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential conflict of interest.","formattedTitle":"Selective dependency of CALR-mutant myeloproliferative neoplasms on TYK2 signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the classical Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), three prominent driver mutations in the genes of janus kinase (JAK) 2, calreticulin (\u003cem\u003eCALR\u003c/em\u003e) and the thrombopoietin (TPO) receptor (\u003cem\u003eMPL\u003c/em\u003e gene\u003cem\u003e/\u003c/em\u003eTPOR protein) are described \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The JAK2V617F mutation occurs most frequently (95% of PV patients, 50\u0026ndash;60% of ET and PMF patients), while \u003cem\u003eCALR\u003c/em\u003e frameshift mutations account for 25\u0026ndash;35% and \u003cem\u003eMPL\u003c/em\u003e mutations for 5\u0026ndash;10% of ET and PMF cases, respectively.\u003c/p\u003e \u003cp\u003eThese driver mutations activate JAKs, explaining the clinical efficacy of the JAK1/2 tyrosine kinase inhibitor (TKI) ruxolitinib \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. JAKs associate with various receptors, including the interferon-α receptor (IFNAR). Upon ligand binding to IFNAR, activated TYK2 and JAK1 phosphorylate signal transducer and activator of transcription (STAT) proteins\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile JAK1 is associated with IFNAR2\u003csup\u003e8\u003c/sup\u003e, TYK2 is associated with IFNAR1 \u003csup\u003e9\u003c/sup\u003e. Besides its role in IFNα signaling, TYK2 activation is involved in cytokine signaling of the IL-10/IL-20, IL-12 and IL-6/gp130 families \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and is implicated in autoinflammatory diseases \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, where TYK2 inhibitors have shown efficacy and good tolerability \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The TYK2 inhibitor deucravacitinib (BMS-986165) has demonstrated clinical efficacy and a favorable safety profile in phase II/III psoriasis trials (POETYK PSO1) and is approved for moderate-to-severe plaque psoriasis \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, deucravacitinib decreased pruritus in psoriasis patients by over 70%, a symptom which is also clinically relevant in MPN patients \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eYamaji et al established a TYK2 knockout in a JAK2V617F-positive mouse model and demonstrated that the loss of TYK2 did not affect the development of the MPN phenotype but was essential for IFNα efficacy \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, similar data are missing for CALR-mutant MPN.\u003c/p\u003e \u003cp\u003eHere, we analyzed the role of TYK2 in CALR- vs. JAK2V617F-mutant cells and observed increased TYK2 phosphorylation and enhanced susceptibility to TYK2 inhibition of CALR-mutant cells. Thus, our data provide new evidence for selective vulnerabilities of CALR- but not JAK2V617F-mutant cells, suggesting that TYK2 inhibitors may be suited for the treatment of patients with CALR-mutant MPN through improved mutant cell selectivity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePrimary patient samples\u003c/p\u003e \u003cp\u003ePeripheral blood samples from MPN patients were obtained from the Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation at RWTH Aachen University Hospital after patients\u0026rsquo; written informed consent, as approved by the local ethics committee (EK 127/12). Healthy control samples were obtained from the Department of Transfusion medicine at RWTH Aachen University Hospital after written informed consent (EK099/14). The list of included patient samples and isolation method of peripheral blood mononuclear cells (PBMC) are provided in the Supplementary information (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eColony formation unit (CFU) assay and calculation of clonogenic variant allele frequency (VAF)\u003c/p\u003e \u003cp\u003eExecution of CFU assays and calculation of clonogenic VAF is described in detail in the Supplementary information and has been described previously \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003e32D (murine hematopoietic myeloid precursor cell line) and TF-1 (human erythroleukemic cell line) cells (DSMZ, Braunschweig, Germany) transduced with \u003cem\u003eMPL-HA\u003c/em\u003e (32D\u003csup\u003eMPL\u003c/sup\u003e and TF-1\u003csup\u003eMPL\u003c/sup\u003e) and either empty vector (EV), murine JAK2V617F or human type 1 mutant CALRdel52 and type 2 mutant CALRins5 were previously described \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and cultured in RPMI1640 medium containing 10% FCS, 10% WEHI supernatant as source of IL-3 (32D) or 2 ng/ml GM-CSF (TF-1) and 1% Pen/Strep. Prior to experiments, cells were IL-3- or GM-CSF-starved, respectively, for 24 h.\u003c/p\u003e \u003cp\u003eAntibodies and Reagents\u003c/p\u003e \u003cp\u003eDeucravacitinib (BMS-986165; MedChemExpress, USA) and ruxolitinib (LC Laboratories, USA) were dissolved in DMSO. Murine IFNα, dissolved in ddH2O, was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies used for Western Blotting are listed in table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eCRISPR/Cas9 targeting of \u003cem\u003eTyk2\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTwo strategies were applied to target the \u003cem\u003eTyk2\u003c/em\u003e gene in 32D cells, as described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and the Supplementary information (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-2). Potential off-targets of the CRISPR/Cas9 procedure were excluded by Sanger sequencing (Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell lysates, SDS-Page and Western Blotting\u003c/p\u003e \u003cp\u003ePreparation of whole cell lysates, SDS-Page and Western Blot (WB) were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. ImageJ or GelAnalyzer (ver. 23.1.1) software was used for quantification of protein levels.\u003c/p\u003e \u003cp\u003eMTT assay\u003c/p\u003e \u003cp\u003eAfter washing cells twice with PBS, 3 x 10\u003csup\u003e4\u003c/sup\u003e cells/well were plated in triplicate on 96 well plates. IFNα and deucravacitinib or fedratinib were diluted in cell culture medium to concentrations ranging from 1\u0026ndash;10,000 U/ml and 0.01 to 10 \u0026micro;M, respectively. DMSO served as control. Metabolic activity of the cells was measured using MTT reagent (Sigma-Aldrich) after 72 h of incubation at 37\u0026deg;C at an absorption of 550 nm.\u003c/p\u003e \u003cp\u003eApoptosis staining\u003c/p\u003e \u003cp\u003e2 x 10\u003csup\u003e5\u003c/sup\u003e cells per ml were treated with 5 \u0026micro;M deucravacitinib for a total duration of 48 h. Apoptosis staining was performed with Annexin V-APC and 7-AAD (1:100 dilution; Biolegend). Samples were analyzed with a Gallios flow cytometer (Beckmann Coulter, Krefeld, Germany).\u003c/p\u003e \u003cp\u003eDifferentiation of induced pluripotent stem cells (iPSC) into hematopoietic stem and progenitor cells (HSPC) and Cell Viability Assay\u003c/p\u003e \u003cp\u003eiPSC-derived MPN clones (JAK2V617F het09, CALRdel52 het01 and CALRins5 het04) were differentiated into HSPC using the spin-EB method as described previously \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. On day 14, CD34\u0026thinsp;+\u0026thinsp;HSPCs were enriched by magnetic activated cell sorting (MACS) according to manufacturer\u0026rsquo;s protocol (Miltenyi, Bergisch-Gladbach, Germany). The Cell Viability Assay is described in the Supplementary information.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least three times unless stated otherwise. Statistical differences were calculated using the tests as indicated in the figure legends, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTYK2 is constitutively phosphorylated in CALR- but not in JAK2V617F-mutated cells\u003c/h2\u003e \u003cp\u003ePreviously, our group investigated differences between CALR- and JAK2V617F- mutant cells in their sensitivity to IFNα treatment illustrating differential JAK1 activation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Given the limited data on the role of TYK2, particularly in CALR-mutated MPN, and the close interaction of both TYK2 and CALR with TPOR, we compared the basal activity of TYK2 in JAK2V617F-mutant versus CALRdel52 cells.\u003c/p\u003e \u003cp\u003e32D\u003csup\u003eMPL\u003c/sup\u003e EV, JAK2V617F, or CALRdel52 cells were analyzed for basal phosphorylation of TYK2 (pTYK2). We found pTYK2 in 32\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which was significantly higher in comparison to 32\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F cells. Increased pTYK2 was confirmed in human TF-1\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 in comparison to JAK2V617F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMutated CALR selectively activates signaling via the TPOR, while the JAK2V617F oncogene interacts with a broader array of receptors\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, implying a direct involvement of TPOR in TYK2 activation downstream of mutated CALR. A model based on the structure of the TYK2 FERM-SH2 domain (PDB ID: 4PO6) in complex with TPOR (red) as well as JAK2 FERM-SH2 domain (PDB ID: 6E2Q) was generated and simulated by molecular dynamics (MD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and see Supplementary Methods). Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations were performed on the MD simulations to estimate binding free energies. Our calculations indicate that TYK2 binds TPOR with comparable affinity to JAK2 (ΔG \u0026asymp; \u0026minus;\u0026thinsp;205 kcal/mol vs \u0026minus;\u0026thinsp;209 kcal/mol), supporting that TYK2 contributes functionally to TPOR signaling, alongside JAK2 (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eWe therefore examined whether TPO stimulation activates TYK2, to determine if the elevated basal pTYK2 levels are associated with constitutive TPOR signaling. TPO stimulation induced pTYK2 in 32D\u003csup\u003eMPL\u003c/sup\u003e EV, JAK2V617F and CALRdel52 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), but the extent of induction was least pronounced in CALRdel52 cells. In addition, upon TPO stimulation, pJAK2 and downstream pSTAT3/5 increased in EV and JAK2V617F but not CALRdel52 cells, while basal STAT phosphorylation was similar between JAK2V617F and CALRdel52. JAK2 whole protein expression was higher in JAK2V617F cells due to the ectopic expression of the oncogene. Interestingly, TPO stimulation strongly activated downstream signaling, including pTYK2, in healthy donor platelets, whereas responses in MPN platelets were weaker and highly variable (Fig. S4A). This likely reflects the significantly reduced TPOR and CD41/CD61 surface expression on ET and MF platelets, independent of driver mutation (Fig. S4B).\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate a direct link between TPOR signaling and TYK2 phosphorylation and highlight the potential relevance of pTYK2 in CALRdel52-mutated cells. Furthermore, TPO stimulation mimics basal CALRdel52-activation of TYK2 downstream of TPOR, which can be amplified in JAK2V617F-expressing cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTPOR/TYK2 interaction is crucial for CALRdel52-induced signaling\u003c/h3\u003e\n\u003cp\u003eTo further investigate the necessity of TYK2 activation and its TPOR interaction in 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F and CALRdel52 cells, two knockout strategies using CRISPR/Cas9 were applied, both aiming at the FERM domain, which facilitates the interaction of TYK2 with its respective receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSanger sequencing of the PCR-amplified TYK2 cDNA revealed that CRISPR strategy 1 yielded clones with small and large deletions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-D). In JAK2V617F- and CALRdel52-mutated background, clones were generated lacking TYK2 phosphorylation. Nevertheless, total TYK2 protein could still be observed in all clones. In a second attempt, aiming to completely eliminate TYK2 protein, we applied CRISPR strategy 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We again recovered pTYK2-deficient clones, however, these were confined to JAK2V617F- and not CALR-mutated 32D\u003csup\u003eMPL\u003c/sup\u003e cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-E). Therefore, we hypothesized that the induced genetic changes via strategy 1 were less severe on downstream signaling compared to strategy 2, explaining the absence of strategy 2-derived 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cell clones.\u003c/p\u003e \u003cp\u003eSince TYK2 is a key mediator of IFNAR1/2 signaling and IFNα inhibits 32D cell growth, we assessed the IFNα response of strategy 1 and strategy 2 mutants using an MTT assay. As expected, JAK2V617F strategy 1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and strategy 2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) cells were significantly less sensitive than their parental counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In line with our hypothesis, pTYK2-deficient JAK2V617F clones resulting from strategy 2 showed almost no reduction of viability in response to IFNα and this reduction in response was significantly more pronounced than in strategy 1-derived clones (IFNα 1000 U; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cells were not informative here and remained IFNα-insensitive, as described before \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo confirm the relevance of pTYK2 for TPO downstream signaling in CALR-mutated cells, we stimulated 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F or CALRdel52 parental cells and strategy 1 and strategy 2 clones with TPO. Apart from complete loss of pTYK2, phosphorylation of STAT factors and JAK2 was preserved in strategy 1 JAK2V617F but not CALRdel52 cells, highlighting the importance of TYK2 for TPOR/CALRdel52 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). JAK2V617F strategy 2 cells showed comparable basal downstream signaling to parental JAK2V617F cells, but were less inducible by TPO, supporting our hypothesis of these being more impaired in their downstream signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eNext, the strategy 2 deletion (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA; aa del158-223), observed in 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F cells was modeled on the TYK2-TPOR complex structure, and MD simulations were performed. Increased structural instability of TYK2 mutant del158-223 complex versus TYK2 WT was reflected by fewer hydrogen bonds throughout the simulation at the TYK2-TPOR interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The deletion of aa 158\u0026ndash;223 induced changes in the aa sequence of the F2 domain and conformational changes in the F1 domain and the linker L1, all part of the FERM domain facilitating the receptor interaction \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. MM-GBSA analysis further supported reduced TPOR binding of the TYK2 del158\u0026ndash;223 mutant, which showed a markedly lower predicted affinity than WT TYK2 (ΔG \u0026minus;\u0026thinsp;154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;31.3 vs. \u0026minus;\u0026thinsp;205.2\u0026thinsp;\u0026plusmn;\u0026thinsp;22.4 kcal/mol; Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These data indicate that del158\u0026ndash;223 destabilizes TPOR\u0026ndash;TYK2 interaction, likely explaining the inability to generate CALRdel52 32DMPL clones using CRISPR strategy 2.\u003c/p\u003e\n\u003ch3\u003eCombination of TYK2- and JAK2-selective inhibitors show synergistic effects\u003c/h3\u003e\n\u003cp\u003eGiven that TYK2 was selectively activated in CALR-mutant but not JAK2V617F-mutant 32D\u003csup\u003eMPL\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we analyzed the efficacy of deucravacitinib, a specific TYK2 inhibitor, including 32D MPLW515K cells, hypothesizing that they may respond similarly to CALRdel52 cells upon TYK2 inhibition.\u003c/p\u003e \u003cp\u003eWe confirmed a significant drop of viability of CALRdel52 and MPLW515K but not JAK2V617F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Correspondingly, we observed a significant increase of apoptosis of 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 but not JAK2V617F cells upon deucravacitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Comparable constitutive pTYK2 levels in CALRdel52 and MPLW515K cells were confirmed, which were abolished by deucravacitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In line with these results, pSTAT1, pSTAT3 and pSTAT5 levels were downregulated only in CALRdel52 and MPLW515K but not JAK2V617F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRuxolitinib\u0026rsquo;s efficacy partly relies on its anti-inflammatory JAK1/JAK2 inhibition \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, whereas the more JAK2-selective inhibitors fedratinib and pacritinib may be promising partners for deucravacitinib. We therefore treated 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F and CALRdel52 cells with 0.5 \u0026micro;M deucravacitinib plus increasing doses of fedratinib for 72 h and assessed viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The combination synergistically reduced viability in CALRdel52 but not JAK2V617F cells (coefficient of drug interaction (CDI)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e below 1 considered as synergistic; 0.68 for 0.5 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.5 \u0026micro;M; 0.38 for 0.5 \u0026micro;M and 1 \u0026micro;M).\u003c/p\u003e \u003cp\u003eDownstream signaling was assessed following treatment with 1 \u0026micro;M deucravacitinib, 0.5 \u0026micro;M fedratinib or their combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In JAK2V617F expressing cells, phosphorylation of STAT proteins was not altered upon these low inhibitor concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Like ruxolitinib, fedratinib is a type I TKI leading to a paradoxical hyperphosphorylation of the regulatory tyrosines in JAK2 upon treatment \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 and MPLW515K cells were most sensitive to deucravacitinib and the combination treatment as demonstrated by a strong downregulation of pSTAT3 and pSTAT5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; Fig. S5). Expression of CALRins5 led to phosphorylation of pTYK2, and the signal was ablated upon deucravacitinib treatment, while pSTAT3 and pSTAT5 were less targeted by the combination treatment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEndogenous JAK2 levels define biologic deucravacitinib efficacy\u003c/h2\u003e \u003cp\u003eStrikingly, these 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 cells expressed higher levels of endogenous JAK2 in comparison to CALRdel52 and MPLW515K expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The high levels of endogenous JAK2 protein in CALRins5-expressing cells may be a factor that affect responsiveness to deucravacitinib, given that TYK2 and JAK2 may compete for TPOR binding \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Therefore, we compared the efficacy of deucravacitinib in CALRdel52 vs CALRins5 cells and demonstrated significant differences in sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Deucravacitinib efficiently reduced pTYK2 and downstream signaling in CALRdel52 cells but was less efficient in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 and JAK2V617F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). STAT5 phosphorylation, in particular, remained unaffected, and again overall JAK2 protein levels were higher in CALRins5 cells in comparison to CALRdel52 cells. To confirm the importance of JAK2 protein expression in the differential response to deucravacitinib, we ectopically overexpressed murine JAK2 WT in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and demonstrated no response to the TYK2 inhibitor up to a concentration of 10 \u0026micro;M (Fig. S6A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we performed single cell dilutions of the 32D\u003csup\u003eMPL\u003c/sup\u003e cell line to discriminate clones with high and low JAK2 protein expression (Fig. S6B). Three cell clones each with low or high JAK2 protein levels were stably transduced with CALRins5. Two clones each were treated with deucravacitinib, and clones #2 and #6 were confirmed to harbor low, and clones #3 and #8 high JAK2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In line with our hypothesis, clones #2 and #6 showed reduced pSTAT3, pSTAT5 and pTYK2 levels and strong sensitivity to deucravacitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Fig. S6C). In contrast, clones #3 and #8 showed an inverse response. To analyze clonal advantage during deucravacitinib treatment, mCherry expressing 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 #3 cells were generated (high JAK2), mixed 1:1 with 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 #6 cells (low JAK2) and treated with deucravacitinib for 48 h. Clonal growth was analyzed by discriminating between GFP+ (#6) and GFP+/mCherry+ (#3) cells in flow cytometry. Although 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 #3 cells started out with a slightly lower percentage, #3 cells outcompeted #6 cells in the presence of the TYK2 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), demonstrating that high JAK2 expression may overcome the sensitivity of CALR-mutant cells to TYK2 inhibition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe JAK2, CALRins5, TPOR clone resists deucravacitinib treatment\u003c/h3\u003e\n\u003cp\u003eWhen assessing 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 clones by flow cytometry for GFP and TPOR surface expression, two populations with differential ratios were observed: for #3 and #8, the main population was GFP\u003csup\u003emed\u003c/sup\u003e (corresponds to CALRins5\u003csup\u003emed\u003c/sup\u003e) and TPOR\u003csup\u003ehigh\u003c/sup\u003e. In comparison, the main population in clones #2 and #6 was GFP\u003csup\u003ehigh\u003c/sup\u003e (CALRins5\u003csup\u003ehigh\u003c/sup\u003e) and TPOR\u003csup\u003emed\u003c/sup\u003e (Fig. S7).\u003c/p\u003e \u003cp\u003eWe hypothesized that the GFP\u003csup\u003emed\u003c/sup\u003e (CALRins5\u003csup\u003emed\u003c/sup\u003e) and TPOR\u003csup\u003ehigh\u003c/sup\u003e population is also JAK2\u003csup\u003ehigh\u003c/sup\u003e, correlating with the JAK2 protein levels in CALRins5 #3 and #6 cells confirmed by WB. As both populations are present in both clones to different extents, we treated 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 #3 and #6 with deucravacitinib for 48 h and 96 h and analyzed GFP/TPOR positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, left). Strikingly, in CALRins5 #6, the GFP\u003csup\u003ehigh\u003c/sup\u003e/TPOR\u003csup\u003emed\u003c/sup\u003e (expected to be JAK2\u003csup\u003elow\u003c/sup\u003e) significantly decreased and in CALRins5 #3, the GFP\u003csup\u003emed\u003c/sup\u003e/TPOR\u003csup\u003ehigh\u003c/sup\u003e (expected to be JAK2\u003csup\u003ehigh\u003c/sup\u003e) population increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, right). Hence, the combination of lower CALRins5, high TPOR surface expression and high JAK2 levels showed resistance to TYK2 inhibition and reduced TYK2-dependency.\u003c/p\u003e\n\u003ch3\u003ePhenotypic response to deucravacitinib is driven by the dependence on TPOR but not EPOR\u003c/h3\u003e\n\u003cp\u003eNext, we investigated whether the CALR-specific effects observed in 32D\u003csup\u003eMPL\u003c/sup\u003e cells also translate to primary patient-derived cells. Therefore, isolated PBMC from MPN patients and healthy controls (HC) were seeded in colony-formation assays with and without deucravacitinib (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Colonies were counted 10\u0026ndash;13 d after seeding, and the number of colonies in drug condition was normalized to the untreated condition. Overall, colony growth was significantly reduced upon treatment (Fig. S8A). Grouping by diagnosis revealed a stronger deucravacitinib effect in ET compared with HC and PV, and in PMF compared with PV, with a trend toward greater sensitivity than HC (p\u0026thinsp;=\u0026thinsp;0.0954) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As PV is driven mainly by erythrocytosis and enhanced EPO signaling, whereas ET and PMF are linked to aberrant megakaryopoiesis and constitutive TPO signaling, we next tested TYK2 inhibition in 32D cells expressing either EPOR or TPOR. Consistent with patient data, deucravacitinib reduced metabolic activity significantly less in EPO-dependent than in TPO-dependent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Next, we examined whether the response to deucravacitinib was genotype-dependent and found that colony growth was comparable across driver oncogene genotypes (Supplementary Fig.S8B). However, when analyzing the fraction of mutated colonies, higher sensitivity of CALR-mutant in comparison to JAK2V617F-mutant colonies was confirmed, as demonstrated by a significantly more pronounced reduction of calculated CALR- vs. JAK2-mutant clonogenic VAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; Fig. S8C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm genotype-related effects of TYK2 inhibition, JAK2V617F-, CALRdel52- and CALRins5-mutant induced pluripotent stem cells (iPSC), that we had previously generated from MPN patients \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, were analyzed. To do this, iPSC-derived CD34\u0026thinsp;+\u0026thinsp;HSPC were treated with deucravacitinib (DMSO, 0.5, 1 and 5 \u0026micro;M) for 72 h, and assessed for cell viability. While CALR-mutated cells were sensitive to TYK2 inhibition, showing significantly reduced viability, JAK2V617F-expressing iPSC-derived CD34\u0026thinsp;+\u0026thinsp;HSPC were resistant to deucravacitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eOverall, these data indicate that CALRdel52-mutated cells are sensitive to TYK2 inhibition, and underscore the influence of disease phenotype and the differential involvement of EPOR versus TPOR signaling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUntil now, TYK2 is primarily known for its role in different autoimmune and inflammatory diseases\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Here, we suggest TYK2 to be a novel pharmaceutical target in CALR-mutant MPN.\u003c/p\u003e \u003cp\u003eThe JAK1/JAK2 inhibitor ruxolitinib is approved for the treatment of myelofibrosis and hydroxyurea-resistant or -intolerant PV, and many patients show excellent hematologic responses including reduction of elevated blood counts, splenomegaly and overall symptom burden \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Next to JAK2 and JAK1, ruxolitinib also inhibits TYK2 activity \u003cem\u003ein vitro\u003c/em\u003e, with an IC50 of 19 nM \u003csup\u003e23,43,44\u003c/sup\u003e. As we observed constitutive TYK2 phosphorylation selectively in CALR- vs. JAK2V617F-mutant cells, the efficacy of ruxolitinib on CALR-mutated MPN may at least partly be attributed to TYK2 inhibition.\u003c/p\u003e \u003cp\u003eWhen stimulating TPOR-expressing cell lines, patient- and HD-derived platelets with TPO, TYK2 was strongly phosphorylated, demonstrating the direct activation of TYK2 downstream of TPOR. Interestingly, our structural model and energy analysis of TPOR/TYK2 and TPOR/JAK2 showed a similar ΔG, which suggests that JAK2 and TYK2 bind the receptor with comparable affinity. TPO-induced TYK2 phosphorylation was low in CALRdel52-expressing cells, likely because mutant CALR binds the TPOR extracellular domain, mimicking cytokine engagement and limiting additional TPO-driven signaling \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. These findings suggest that CALRdel52 activates TYK2 by inducing a TPOR intracellular conformation similar to that triggered by TPO binding. This would be in line with Papadopoulos et al, suggesting that mutant CALR effectively replaces TPO as an artificial ligand\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the suggested tetrameric mutant CALR-TPOR architecture contrasts with 1:2 TPO:TPOR cryo-EM structures, described by Tsutsumi et al, highlighting potential structural differences at the intracellular JAK binding sites\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In contrast, JAK2V617F binding to TPOR may promote a distinct C-terminal conformation that favors JAK2/receptor association.\u003c/p\u003e \u003cp\u003eWe aimed to generate TYK2-deficient JAK2V617F- and CALRdel52-mutant 32D\u003csup\u003eMPL\u003c/sup\u003e cells utilizing two different CRISPR/Cas9 knockout strategies (nickase or nuclease). With both strategies, pTYK2-deficient 32D\u003csup\u003eMPL\u003c/sup\u003e JAK2V617F cell lines were successfully established, but in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cells, only strategy 1 was successful. No complete TYK2 knockout was obtained in any of the cell lines. Strategy 2 yielded clones that were most strongly impaired in their downstream signaling when treated with TPO or IFNa, and when analyzed by MD simulations.\u003c/p\u003e \u003cp\u003eDeucravacitinib, a selective TYK2 inhibitor, targets the pseudokinase domain of TYK2, which makes it more specific than ruxolitinib \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Inhibition of TYK2 had a significantly stronger effect on CALRdel52- and MPLW515K-expressing cells compared to JAK2V617F-mutant cells, demonstrating the relevance of TYK2 for TPOR-driven signaling in these cells. Oncogenic signaling of CALR mutations relies on TPOR \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, whereas JAK2V617F also interacts with EPOR and CSF3R \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. TYK2 has been reported to associate with TPOR and, like JAK2, regulate its surface expression \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Reduced TYK2 activity or binding affinity could thus lower TPOR surface levels. Likewise, TYK2 deficiency reduces IFNAR1 surface expression by driving it into perinuclear compartments \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Nevertheless, TYK2 inhibition by deucravacitinib did not alter TPOR surface expression in JAK2V617F- or CALR-mutant 32D\u003csup\u003eMPL\u003c/sup\u003e cells (data not shown).\u003c/p\u003e \u003cp\u003eTo our surprise, deucravacitinib did not reduce cell viability of 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 expressing cells, and it showed less effect on downstream signaling in these cells. The reduced response to TYK2 inhibition was attributable to higher endogenous JAK2 protein levels in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 clones compared with CALRdel52 clones. We confirmed that the 32D\u003csup\u003eMPL\u003c/sup\u003e CALRins5 JAK2\u003csup\u003ehigh\u003c/sup\u003e clones were less sensitive to TYK2 inhibition and outcompeted JAK2\u003csup\u003elow\u003c/sup\u003e cells in co-culture with deucravacitinib. In addition, a dominant surface TPOR\u003csup\u003ehigh\u003c/sup\u003e/GFP\u003csup\u003emed\u003c/sup\u003e population was present in deucravacitinib-resistent clones, while a TPOR\u003csup\u003emed\u003c/sup\u003e/GFP\u003csup\u003ehigh\u003c/sup\u003e population was dominant in deucravacitinib-sensitive clones. All clones showed a similar basal pTYK2 band in WB. Hence, pTYK2 was not the main driver of TPOR surface expression but rather JAK2 and the amount of CALRins5 expression (indicated by GFP). Chachoua and colleagues described reduced maturation of TPOR in mutant CALR expressing cells \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, thereby reducing surface trafficking, which may then depend on the amount of mutant CALR per cell. Overall, in patients with CALR mutations, particularly with TPOR\u003csup\u003emed\u003c/sup\u003e JAK2 WT\u003csup\u003elow\u003c/sup\u003e CALRmut\u003csup\u003ehigh\u003c/sup\u003e, the HSPC population may especially benefit from deucravacitinib treatment. However, the selection of JAK2\u003csup\u003ehigh\u003c/sup\u003e clones under deucravacitinib treatment highlights the importance to target simultaneously JAK2 and TYK2. Combination of deucravacitinib and fedratinib induced synergistic effects in 32D\u003csup\u003eMPL\u003c/sup\u003e CALRdel52 cells. Although combination of different JAK inhibitors has been discussed, particularly in MPN and autoimmune diseases, studies are currently still preclinical or entering early clinical testing \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Special caution would be required due to overlapping immunosuppression and a potential increased risk of infection or cytopenia.\u003c/p\u003e \u003cp\u003eExperiments on primary MPN patient material confirmed selective sensitivity to TYK2 inhibition in CALR-mutant cells from patients with ET and PMF, but not those from patients with JAK2V617F-mutant cells and those with PV harboring a JAK2 mutation. Mechanistically, we linked the selective TYK2 dependence to those MPN, where the TPOR (and not EPOR) is the main driver of cell proliferation. Importantly, we showed that TYK2 inhibition led to a significantly stronger decrease in the calculated VAF in CALR- vs. JAK2V617F-mutant clonogenic cells and even more immature CD34\u0026thinsp;+\u0026thinsp;iPSC-derived HSPC.\u003c/p\u003e \u003cp\u003eIn summary, our data provides the rationale for a new therapeutic approach for patients with MPN, particularly those harboring CALR mutations. Deucravacitinib is approved for the treatment of patients with psoriasis, demonstrating good clinical activity and acceptable toxicity\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, with no relevant anemia or thrombocytopenia. As TYK2 plays an important role in the persistence of JAK2V617F-positive cells during ruxolitinib therapy \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, it might be interesting to analyze the effects of a double treatment with JAK2- and TYK2-specific inhibitors. In MPN patients suffering from (ro)pegIFNα-induced pruritus, deucravacitinib, which has been shown to reduce itch in psoriasis patients by over 70% in two weeks, could be used to treat this pruritus \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In those MPN cases of concomitant TKI and IFNα therapy, TYK2 inhibition may partly counteract the IFNα effects. However, this was not seen in the clinical setting for ruxolitinib and pegylated IFNα \u003csup\u003e60\u003c/sup\u003e, possibly due to differential kinetics of pegIFNα and the JAK inhibitor \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Finally, deucravacitinib may be an ideal combination partner for the newly-developed mutant-CALR-specific antibodies, which are currently in clinical trials (NCT05936359 and NCT06150157).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was in part funded by research grants to SK from the \u003cem\u003eDeutsche Forschungsgemeinschaft\u0026nbsp;\u003c/em\u003e(German Research Foundation) (DFG KO 2155/6-1, project number AOBJ 636363) (DFG KO 2155/8-2, project number AOBJ: 680695)\u0026nbsp;and by funds from the German Research Foundation as part of the Clinical Research Unit CRU 344 to S.K. (KO2155/7-1, project number 428858786) and N.C. (CH1509/1-1) as well as the \u003cem\u003eDeutsche José Carreras Leukämie-Stiftung\u003c/em\u003e (DJCLS R 16/2017). This work was supported by RWTH Aachen University through Seed Funds Project OPSF702 of G.R. and N.C.. The work was partly performed in the Core Facility Flow Cytometry, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University. Biomaterial samples were provided by the RWTH centralized Biomaterial Bank Aachen (RWTH cBMB, Aachen, Germany) in accordance with the regulations of the biomaterial bank and the approval of the ethics committee of the medical faculty, RWTH Aachen.\u0026nbsp;G.R. and A.P. acknowledge the Federal Ministry of Education and Research (BMBF) and the state of North Rhine-Westphalia as part of the NHR Program.\u0026nbsp;Part of this work was generated within the medical thesis works of RL, LS, and CW. MK is supported by a postdoctoral fellowship from de Duve Institute and by a Fonds Speciaux de recherches fellowship of UCLoouvain.\u0026nbsp;Funding to SNC is acknowledged from Ludwig Institute for Cancer Research, Fondation contre le cancer, Salus Sanguinis and Fondation “Les avions de Sébastien”, projets Action de recherché concertée (ARC) 16/21-073, Projet de recherche FNRS n°T.0043.21 and WelBio F 44/8/5 - MCF/UIG – 10955.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSteffen Koschmieder received research funding from Geron, Janssen, AOP Pharma, and Novartis; received consulting fees from Pfizer, Incyte, Ariad, Novartis, AOP Pharma, Bristol Myers Squibb, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, GSK, Sierra Oncology, and PharmaEssentia; received payment or honoraria from Novartis, BMS/Celgene, Pfizer, AstraZeneca, and iOMEDICO; received travel/accommodation support from Alexion, Novartis, Bristol Myers Squibb, Incyte, AOP Pharma, CTI BioPharma, Pfizer, Celgene, Janssen, Geron, Roche, AbbVie, GSK, Sierra Oncology, Kartos, AstraZeneca, Protagonist, and iOMEDICO; had a patent issued for a BET inhibitor at RWTH Aachen University; participated on advisory boards for Pfizer, Incyte, Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, GSK, Sierra Oncology, and PharmaEssentia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK, CW and RL designed the research, performed the experiments, analyzed the data, and wrote the manuscript. BJ, AP, JG, SG and LS performed experiments and analyzed the data. AS, ST, KP, MAST and AG performed experiments. KK and JS collected patient data. MAST, CP and SNC designed research, analyzed the data and corrected the manuscript. SK and NC designed the research, analyzed the data, and corrected the manuscript. SK collected patient samples. All authors approved the final version of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGreenfield G, McMullin MF, Mills K. 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Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation. \u003cem\u003eBlood\u003c/em\u003e 2023; 142: 1818\u0026ndash;1830.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsutsumi N, Masoumi Z, James SC, Tucker JA, Winkelmann H, Grey W \u003cem\u003eet al.\u003c/em\u003e Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis. \u003cem\u003eCell\u003c/em\u003e 2023; 186: 4189\u0026ndash;4203.e22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWrobleski ST, Moslin R, Lin S, Zhang Y, Spergel S, Kempson J \u003cem\u003eet al.\u003c/em\u003e Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165. \u003cem\u003eJ Med Chem\u003c/em\u003e 2019; 62: 8973\u0026ndash;8995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElf S, Abdelfattah NS, Chen E, Perales-Pat\u0026oacute;n J, Rosen EA, Ko A \u003cem\u003eet al.\u003c/em\u003e Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. \u003cem\u003eCancer Discov\u003c/em\u003e 2016; 6: 368\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAraki M, Yang Y, Masubuchi N, Hironaka Y, Takei H, Morishita S \u003cem\u003eet al.\u003c/em\u003e Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. \u003cem\u003eBlood\u003c/em\u003e 2016; 127: 1307\u0026ndash;1316.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu X, Levine R, Tong W, Wernig G, Pikman Y, Zarnegar S \u003cem\u003eet al.\u003c/em\u003e Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2005; 102: 18962\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Li\u0026ntilde;ares B, Watson SP. Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation. \u003cem\u003eBiochem J\u003c/em\u003e 1996; 316 (Pt 1: 93\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrachman JG, Millett KM, Kaushansky K. Thrombopoietin signal transduction requires functional JAK2, not TYK2. \u003cem\u003eJ Biol Chem\u003c/em\u003e 1999; 274: 13480\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRagimbeau J, Dondi E, Alcover A, Eid P, Uz\u0026eacute; G, Pellegrini S. The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression. \u003cem\u003eEMBO J\u003c/em\u003e 2003; 22: 537\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu R-I, Marty C \u003cem\u003eet al.\u003c/em\u003e Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. \u003cem\u003eBlood\u003c/em\u003e 2016; 127: 1325\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalpaz M, Gerds AT, Lyons R, Langmuir P, Hunter D, Lamothe B \u003cem\u003eet al.\u003c/em\u003e A phase 2 study of itacitinib alone or in combination with low-dose ruxolitinib in patients with myelofibrosis. \u003cem\u003eLeuk Res\u003c/em\u003e 2025; 155: 107732.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuepper MK, B\u0026uuml;tow M, Herrmann O, Ziemons J, Chatain N, Maurer A \u003cem\u003eet al.\u003c/em\u003e Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling. \u003cem\u003eLeukemia\u003c/em\u003e 2019. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41375-019-0427-7\u003c/span\u003e\u003cspan address=\"10.1038/s41375-019-0427-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoppikar P, Bhagwat N, Kilpivaara O, Manshouri T, Adli M, Hricik T \u003cem\u003eet al.\u003c/em\u003e Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy. \u003cem\u003eNature\u003c/em\u003e 2012; 489: 155\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026oslash;rensen AL, Mikkelsen SU, Knudsen TA, Bj\u0026oslash;rn ME, Andersen CL, Bjerrum OW \u003cem\u003eet al.\u003c/em\u003e Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study. \u003cem\u003eHaematologica\u003c/em\u003e 2020; 105: 2262\u0026ndash;2272.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1. MM-GBSA calculations of the predicted binding free energy (\u0026Delta;G) along the simulations of TPOR in complex with JAK2 WT, TYK2 WT, TYK2 del158-223.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 50.3012%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComplex with TPOR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6988%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003eG\u003cem\u003e\u0026nbsp;\u003c/em\u003ein kcal/mol (mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 50.3012%;\"\u003e\n \u003cp\u003eJAK2 WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6988%;\"\u003e\n \u003cp\u003e-209.75 \u0026plusmn; 28.74\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 50.3012%;\"\u003e\n \u003cp\u003eTYK2 WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6988%;\"\u003e\n \u003cp\u003e-205.17 \u0026plusmn; 22.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 50.3012%;\"\u003e\n \u003cp\u003eTYK2 del158-223\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6988%;\"\u003e\n \u003cp\u003e-154.76 \u0026plusmn; 31.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":true,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8425696/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8425696/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMyeloproliferative neoplasms (MPN) are driven by the oncoproteins JAK2V617F, mutant calreticulin (CALR), and mutant thrombopoietin receptor (TPOR), all of which activate JAK/STAT signaling. While JAK2 signaling is engaged in all MPNs, TYK2 is dispensable for JAK2V617F-driven disease. Here, we hypothesized a distinct role for TYK2 in CALR-mutant driven MPN. We found constitutive TYK2 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-expressing cells. Modelling of JAK2/TPOR vs. TYK2/TPOR interaction confirmed similar binding affinities of both kinases to the receptor, which was more relevant in CALRdel52-positive cells. The TYK2 inhibitor deucravacitinib reduced viability and STAT3/5 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-mutant cells, with enhanced efficacy when combined with the JAK2-selective inhibitor fedratinib. Cellular response correlated with JAK2 protein abundance, as CALRins5 JAK2\u003csup\u003ehigh\u003c/sup\u003e clones outcompeted JAK2\u003csup\u003elow\u003c/sup\u003e clones upon TYK2 inhibition. In primary samples, deucravacitinib significantly suppressed colony growth in ET and PMF but not PV, and selectively reduced CALR- but not JAK2V617F-mutant allele burden. Similarly, CALR-mutant patient-specific iPSC-derived CD34\u003csup\u003e+\u003c/sup\u003e progenitors were more sensitive to TYK2 inhibition than their JAK2V617F counterparts. These findings identify TYK2 as a selective vulnerability in CALR-mutant MPN and support combined TYK2/JAK2 inhibition strategies to overcome JAK2-dependent resistance.\u003c/p\u003e","manuscriptTitle":"Selective dependency of CALR-mutant myeloproliferative neoplasms on TYK2 signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-07 08:51:53","doi":"10.21203/rs.3.rs-8425696/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-05T14:49:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-03T05:32:40+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-25T12:09:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-15T23:35:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-06T15:47:54+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-06T15:18:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-06T11:51:14+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-06T10:01:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T11:27:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T11:17:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2025-12-22T13:21:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d7c7fdfd-1238-4477-8ddd-c87a469d281a","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":60662617,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Myeloproliferative disease"},{"id":60662618,"name":"Biological sciences/Stem cells/Haematopoietic stem cells"},{"id":60662619,"name":"Biological sciences/Cell biology/Cell signalling"}],"tags":[],"updatedAt":"2026-04-16T19:25:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 08:51:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8425696","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8425696","identity":"rs-8425696","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}