JAK2 V617F Myeloproliferative Neoplasms Support Parallel Evolution of Independent Leukemic Clones

JAK2V617F Myeloproliferative Neoplasms Support Parallel Evolution of Independent Leukemic Clones | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results JAK2 V617F Myeloproliferative Neoplasms Support Parallel Evolution of Independent Leukemic Clones View ORCID Profile Tyler M. Parsons , Aishwarya Krishnan , Infencia Xavier Raj , Andrew L. Young , David R. O’Leary , Jason Arand , Maggie Cox , Stephen T. Oh , View ORCID Profile Grant A. Challen doi: https://doi.org/10.1101/2025.09.23.678057 Tyler M. Parsons 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tyler M. Parsons Aishwarya Krishnan 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Infencia Xavier Raj 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andrew L. Young 2 Division of Hematology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site David R. O’Leary 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jason Arand 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maggie Cox 2 Division of Hematology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen T. Oh 2 Division of Hematology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Grant A. Challen 1 Division of Oncology, Department of Medicine, Washington University School of Medicine ; St. Louis, MO, USA , 63110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Grant A. Challen For correspondence: grantchallen{at}wustl.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Myeloproliferative neoplasms (MPNs) are hematological diseases predominantly driven by the JAK2 V617F mutation. Progression from chronic-phase MPN to secondary acute myeloid leukemia (sAML) is a severe complication that dramatically worsens disease prognosis. While progression to sAML is classically linked to MPN clones acquiring additional mutations, the absence of JAK2 V617F in some cases of post-MPN sAML cases suggests alternative mechanisms of transformation. Utilizing patient samples and in vivo modeling, we establish that leukemic clones can emerge independently of JAK2 -mutant cells and undergo positive selection in the pro-inflammatory MPN environment, leading to parallel disease evolution. Genetic and pharmacological inhibition of IL-12 and TNFα mitigates this competitive advantage. Our data establish a new paradigm and show that disease progression in MPN can arise from parallel acute myeloid leukemia (pAML) clones. Main Text Myeloproliferative neoplasms (MPNs) are a group of chronic hematological diseases driven by the acquisition of somatic mutations, predominantly JAK2 V617F , in hematopoietic stem cells (HSCs)( 1 – 3 ). MPNs are characterized by the aberrant and unregulated proliferation of one or more myeloid lineages, resulting in the overproduction of mature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB)( 4 – 6 ). This sustained overproduction manifests as polycythemia vera (PV; excess erythrocytes), essential thrombocythemia (ET; excess platelets), or myelofibrosis (MF; BM fibrosis). Consequently, patients face increased risks of blood viscosity and clotting, organ enlargement, joint pain and swelling, BM scarring, and are at-risk for progression to more aggressive disease states. The transformation of chronic-phase MPN to secondary acute myeloid leukemia (sAML) is a severe complication traditionally linked to the acquisition of additional mutations in the MPN driver clone in genes encoding epigenetic regulators (e.g. TET2, DNMT3A, ASXL1) , tumor suppressors (e.g. TP53, JARID2) and signaling molecules (e.g. N / K - RAS) ( 7 – 10 ). However, the JAK2 V617F driver mutation is occasionally absent in sAML that transforms from antecedent JAK2 -mutant MPNs, which suggests alternative mechanisms of disease evolution( 11 ). Proposed explanations for the absence of the JAK2 V617F mutation at the sAML stage include somatic reversion and loss of heterozygosity, but an underexplored possibility is that sAML originates from independent clones that evolve in parallel to the MPN during the natural history of clonal hematopoiesis (CH)( 12 , 13 ). Here, we establish that pre-leukemic clones can arise independently of the MPN and outcompete the JAK2 V617F -mutant cells to manifest disease progression, challenging the traditional dogma of post-MPN sAML evolution. We leveraged a series of primary patient samples (Table S1) and pre-clinical models to investigate the growth and evolution of parallel clones in an MPN background. We demonstrate that independent clones carrying TET2 and TP53 mutations are positively selected by MPN-derived pro-inflammatory cytokines such as IL-12 and TNFα. Importantly, we demonstrate these mechanisms are amenable to targeted therapy as genetic and pharmacological inhibition of IL-12- and TNFα-signaling ameliorates the competitive advantage of TET2 -mutant cells in the presence of JAK2 V617F -mutant MPN. Collectively, these data demonstrate that JAK2 V617F -mutant cells condition an environment that confers a selective advantage for the parallel growth of independent clones in the background of existing MPN. In such cases, the more accurate diagnosis is two independent diseases evolving in the same patient – the primary MPN and a parallel acute myeloid leukemia (pAML). Improved understanding of the phylogeny of MPN disease evolution may offer new clinical opportunities for these patients who currently have very limited treatment options. Leukemic Clones Can Arise Independently and Undergo Positive Selection in a Background of MPN To investigate the clonal trajectory post-MPN sAML, we performed single cell genomic analysis for three paired patient samples (2 MF; 1 PV) who progressed from a JAK2 V617F -mutant MPN to a JAK2 -negative sAML as defined by clinical sequencing (Fig. S1A) . Single-cell sequencing data from purified CD34 + cells for two patients (MF UPN:950899; PV UPN:374024) demonstrated that mutations detected at the sAML stage were not detected within the JAK2 -mutant clones at the MPN stage ( Fig. 1A ). Despite clinical genomics defining MF UPN:638574 as JAK2 -negative sAML, single cell analysis of purified CD34 + cells clearly showed the leukemic mutations were acquired in the founding MPN clone ( Fig. 1A ) . This was confirmed by droplet digital PCR (ddPCR) wherein the JAK2 V617F mutation was detected at both the MPN and the sAML stages (Fig. S1B) . While these data show there are multiple routes to transformation of post-MPN sAML, the single cell genomics clearly show that the leukemia-initiating mutations are not always present within the JAK2 -mutant clones and can arise independently and outcompete the MPN cells to drive transformation. Download figure Open in new tab Fig. 1. Leukemic Clones Can Arise Independently and Undergo Positive Selection in a Background of MPN. (A) Clonal hierarchy visualization of three paired (MPN and sAML) patient samples resolved by single-cell genomic sequencing with the x-axis expressed as time and the y-axis displaying proportionate prevalence of each clone within the population. (B) ex vivo competition assay showing VAF of engineered AAVS1, TET2 , and TP53 mutations in cord blood CD34 + cells in co-culture with CD34 + cells from JAK2 V617F PV or MF patients or healthy donor (HD) BM. Given this finding, we developed an ex vivo competition system to quantify the proliferation of independent clones in the presence of MPN cells utilizing CRISPR/Cas9 editing. 1×10 5 CD34 + cells from JAK2 -mutant MPN patients or healthy donor (HD) BM were co-cultured for 12-days with 2×10 4 cord blood (CB)-derived CD34 + cells nucleofected with gRNAs targeting AAVS1, TET2 or TP53 . CRISPR edited alleles were tracked via targeted genomic sequencing at 6-day intervals and compared to initial values 24-hours post-nucleofection. In the presence of MPN patient CD34 + cells, TET2 - and TP53 -mutant clones expanded significantly more compared to co-culture of the same clones with HD BM control CD34 + cells ( Fig. 1B ) . Interestingly, PV patient cells supported the growth of both TET2 - and TP53 -mutant cells moreso than MF patient cells, a finding consistent with clinical reports that the JAK2 -mutant MPN to JAK2 wild-type sAML trajectory is more common in PV patients( 11 , 13 ). JAK2 V617F -Mutant MPN Accelerates Expansion of Independent TET2 - and TP53 -Mutant Clones To explore competition dynamics between MPN cells and independent clones, we leveraged our MPN patient derived xenograft (PDX) system( 14 ). TET2 and TP53 mutations were engineered into CB-derived CD34 + cells using CRISPR/Cas9 homology-directed repair (HDR) to introduce TET2 1216* and TP53 R248Q knock-in (KI) mutations into CB-derived CD34 + cells (Fig. S2) . These mutations were chosen from the limited literature describing TET2 and TP53 mutations as the most common variants in JAK2 -negative post-MPN sAML( 15 , 16 ). A single-stranded oligo donor nucleotide (ssODN) was designed to introduce a silent mutation in the inert AAVS1 locus to serve as a trackable negative control genetic barcode. As most TET2 and TP53 mutations in myeloid malignancies lead to loss of function( 17 – 22 ), indels resulting from CRISPR/Cas9 cutting but failed ssODN directed repair were additionally tracked. These edits should also provide the cells with a competitive advantage and increase the clonal complexity within a given experiment. PDX models were established by co-transplanting 2.0×10 4 cells from each of the AAVS1, TET2 and TP53 nucleofected populations into NSGS immunodeficient mice with 1.0×10 5 CD34 + cells derived from either HD BM confirmed to be JAK2 V617F -negative by ddPCR (control; n=4), JAK2 -mutant MF (n=4), or JAK2 -mutant PV (n=4) patients (Table S1) . Flow cytometric analysis was performed to confirm PB lineage reconstitution (Fig. S3) , and PDX models supported robust engraftment of human cells ( Fig. 2A ) in the BM ( Fig. 2B ). Notably, mice engrafted with PV patient cells supported significant expansion of human HSCs (mCD45-hCD45+ LINEAGE-CD34+ CD38-CD45RA-CD90+) in the BM ( Fig. 2C ). PDXs recapitulated classical phenotypes of the patients from which they were derived (WBC, HCT, splenomegaly; Fig. 2D-F ) and served as a relevant platform to observe how different MPN subtypes support the expansion of independent TET2 - and TP53 -mutant clones. Targeted genomic sequencing was performed on hCD45+ cells isolated from the BM of recipient mice at the conclusion of each independent transplant (16-weeks) to quantify gRNA-mediated variant allele fraction (VAF). These data were compared to pre-transplant values to analyze the growth dynamics of parallel clones isolated from a JAK2 V617F -mutant MPN-derived PDX host in comparison to the same pool of edited cells in a HD control host. MF and PV patient cells supported the growth of TET2- and TP53- mutant clones significantly more than HD control BM across four separate experiments established with independent starting MPN patient material ( Fig. 2G ) . Notably, cohorts established from PV patient cells exhibited the highest expansion of independent clones, particularly for TET2 -mutant cells, mirroring results from the ex vivo experiment. These data highlight the robust expansion capacity of TET2 - and TP53 -mutant clones within JAK2 V617F -mutant MPN environments in vivo . Download figure Open in new tab Fig. 2. JAK2 V617F -Mutant MPN Accelerates Expansion of Independent TET2 - and TP53 - Mutant Clones. Representative flow cytometry plot depicting human cell engraftment in BM of NSGS mice. Quantification of overall hCD45+ BM engraftment, (C) human HSC number, (D) white blood cell count, (E) hematocrit and (F) spleen weights compiled across PDX experiments. (G) Relative fold change of CRISPR modified reads in hCD45+ BM cells 16-weeks post-transplant compared to day 0 values of tracked AAVS1, TET2 , and TP53 engineered mutations in cord-blood CD34 + cells (n= 12 recipient mice sourced from 4 separate donors for HD, PV, and MF). ** p ≤0.01, **** p ≤0.0001 Low Burden of Jak2 V617F -Mutant Cells Specifically Supports Parallel Expansion of Tet2 -Mutant Clones With the finding that independent TET2 and TP53 clones display a significant growth advantage in a JAK2 V617F -mutant environment, we established murine chimera models to provide a platform for mechanistic studies. These systems allow for precise control of the burden of each mutant cell population at the start of each transplant, facilitating a more detailed study of clone behavior and interaction than afforded by PDX models. We utilized mice with inducible expression of Jak2 V617F in the hematopoietic system (Vav-Cre; Jak2 V617F/+ = “ Jak2 V617F ”), which generate a PV-like phenotype, or wild-type (WT) control mice as a host background and Tet2 heterozygous loss-of-function (Vav-Cre; Tet2 fl/+ = “ Tet2 Δ/+ ”) or Tp53 heterozygous knock-in (Tp53 R172H/+ ) mice as the competing test populations. 2.5×10 6 BM cells from either Jak2 V617F or WT control mice (CD45.2) were mixed with 5×10 5 BM cells from either Tet2 Δ/+ , Tp53 R172H/+ , or WT test BM (CD45.1/2) and transplanted into lethally irradiated recipients (CD45.1) to produce a starting fraction of “test” BM of approximately 15%. Flow cytometric analysis was performed to evaluate PB lineage reconstitution (Fig. S4) . By 16-weeks post-transplant, Jak2 V617F -mutant host cells significantly supported expansion of both Tet2 Δ/+ and Tp53 R172H/+ test populations in the PB compared to a WT control host background ( Fig. 3A-B ). However, the WT test population also exhibited an engraftment increase in the presence of Jak2 V617F -mutant cells ( Fig. 3B ) . This engraftment advantage was largely restricted to peripheral lymphoid cells, consistent with prior reports that JAK2 V617F mutations confer lymphoid deficiency. Within the PB myeloid compartment in a Jak2 V617F -mutant background, the WT test population exhibited significantly reduced chimerism compared to Tet2 Δ/+ and Tp53 R172H/+ test populations ( Fig. 3C-D ) . BM analysis 18-weeks post-transplant revealed a similar competitive advantage of Tet2 Δ/+ and Tp53 R172H/+ cells in a Jak2 V617F -mutant background ( Fig. 3E ) . Interestingly, the effect was most pronounced in the hematopoietic stem/progenitor cell (HSPC; c-Kit+ Sca-1+ Lineage-“KSL”) compartment rather than the most primitive long-term hematopoietic stem cells (HSCs; KSL CD48-CD150+) ( Fig. 3F-G ) . Download figure Open in new tab Fig. 3. Low Burden of Jak2 V617F -Mutant Cells Specifically Supports Parallel Expansion of Tet2 -Mutant Clones. (A, B) Peripheral blood engraftment of WT control, Tet2 Δ/+ and Tp53 R172H/+ test cells in (A) WT or (B) a Jak2 V617F host BM. (C, D) PB myeloid (Gr-1 + Cd11b + ) engraftment of WT control, Tet2 Δ/+ and Tp53 R172H/+ test cells in (C) WT or (D) Jak2 V617F host BM. (E-G) Quantification of WT, Tet2 Δ/+ , Tp53 R172H/+ test cell (E) BM engraftment, (F) HSPC (Lineage - Sca-1 + c-Kit + ) number and (G) HSC (Lineage - Sca-1 + c-Kit + CD48 - CD150 + ) number in the BM of recipient mice 18-weeks post-transplant (n =10 / group). (H-J) Quantification of WT control and Tet2 Δ/+ cell (H) PB engraftment, (I) BM engraftment and (J) HSPC numbers in recipient mice transplanted with indicated fractions of Jak2 V617F BM input cells (n = 5 / group). (K-N) Quantification of WT control, Tet2 Δ/+ , Tp53 R172H/+ test cell (K) PB engraftment, (L) BM engraftment, (M) HSPC numbers and (N) HSC numbers in recipient mice transplanted with 50% Jak2 V617F BM input (n= 5-9 / group). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 As Tet2 -mutant clones demonstrated an enhanced growth advantage in a Jak2 V617F -environment compared to Tp53 -mutant clones in both PDX and murine chimeras, we aimed to determine the minimum Jak2 V617F -mutant cell burden required to support parallel Tet2 -mutant clone expansion. A titration experiment was performed wherein BM cells from Jak2 V617F mice were diluted at predetermined ratios with WT BM while maintaining a fixed Tet2 Δ/+ cell input (15%). There was a threshold effect between starting Jak2 V617F cell input and the conferred competitive advantage of Tet2 Δ/+ cells with a ∼35% Jak2 V617F mutant cell burden being the threshold needed to support robust expansion of Tet2 Δ/+ cells ( Fig. 3H-J ) . Based on these data, we established chimeras with 50% Jak2 V617F -mutant BM, 35% WT support BM, and 15% Tet2 Δ/+ , Tp53 R172H/+ or WT test BM to determine if this was also sufficient to induce outgrowth of parallel Tp53 -mutant cells. As anticipated, a 50% starting Jak2 -input supported positive selection of Tet2 Δ/+ cells in the PB, BM, and HSPC compartments. However, this Jak2 V617F -mutant burden did not support engraftment of Tp53 R172H/+ or WT cells unlike the higher (85%) Jak2 V617F -mutant cell input ( Fig. 3K-M ) . These findings highlight a specific interaction between Jak2 V617F -mutant MPN cells and independent Tet2 -mutant clones. IL-12 and TNFα Drive Expansion of Tet2 -Mutant Clones in a Jak2 V617F -Mutant Environment As our data show a particularly strong selection of independent Tet2 -mutant clones in an MPN background, we focused mechanistic studies to define this relationship. As the selective advantage for Tet2 Δ/+ cells in and MPN background was most evident at the level of HSPCs, gene expression profiling was performed by RNA-sequencing analysis of Tet2 Δ/+ HSPCs (CD45.1/2+ c-Kit+ Sca-1+ Lineage-) isolated from either WT or Jak2 V617F -mutant hosts 18-weeks post-transplant. Analysis of differentially expressed genes (DEGs; Fig. 4A ) demonstrated that Tet2 Δ/+ HSPCs from a Jak2 V617F -mutant environment exhibited a pronounced proliferation bias, exemplified by upregulation of Mki67 , and increased expression of pro-myeloid differentiation genes such as Elane, Mpo , and Cebpe . The inflammatory response was also heightened with elevated levels of S100a8 and S100a9 which function as key promoters of myeloid inflammation and are routinely upregulated in HSPCs in the contexts of myeloid skewing and chronic inflammatory states( 23 ). This myeloid differentiation bias occurred at the expense of lymphoid priming marked by decreased expression of Dntt and Lck , essential genes for lymphoid lineage commitment. Genes classically associated with HSC identity, such as Hoxa10, Fgd5, Vldlr, Hlf , and Mecom , were also downregulated in Tet2 Δ/+ HSPCs from a Jak2 V617F -mutant environment ( Fig. 4B ) , suggesting these cells were being pushed towards proliferation at the expense of self-renewal. Collectively, these findings indicate that a Jak2 V617F -mutant environment drives Tet2 -mutant HSPCs toward a proliferative, pro-myeloid, inflammatory phenotype. We next interrogated whether the gene expression changes observed in Tet2 Δ/+ HSPCs from a Jak2 V617F -mutant environment were due to specific effects on Tet2 Δ/+ cells and not a general effect for any HSPCs exposed to MPN cells by RNA-seq comparison of Tet2 Δ/+ and WT HSPCs isolated from a Jak2 V617F -mutant environment. Differential expression analysis confirmed that the transcriptomic alterations previously identified in Tet2 Δ/+ HSPCs remained dysregulated in Tet2 Δ/+ HSPCs compared to WT HSPCs from a Jak2 V617F -mutant environment (Fig. S5) . These findings demonstrate that the identified gene dysregulation is unique to Tet2 -mutant cells within a Jak2 V617F -mutant milieu and not a generalized feature of HSPCs exposed to MPN cells. Download figure Open in new tab Fig. 4. IL-12 and TNFα Drive Expansion of Tet2 -Mutant Clones in a Jak2 V617F -Mutant Environment (A) Volcano plot showing differentially expressed genes between Tet2 Δ/+ HSPCs from a Jak2 V617F environment compared to Tet2 Δ/+ HSPCs from a WT environment. (B) LogFC values for Tet2 Δ/+ HSPCs DEGs from indicated pathways. (C) Volcano plot of gene set over-representation analysis (ORA) identifying IL-12 signaling as the most significantly enriched pathway in Tet2 Δ/+ HSPCs from a Jak2 V617F environment. (D-E) Serum cytokine levels (normalized to WT control chimeras) of (D) IL-12 and (E) TNFα from murine mixed chimeras (n= 9-15 / group). ( F ) Serum cytokine fold change (normalized to HD control mice) for PV and MF PDXs plotted against relative increase in VAF of individually tracked TET2 mutations engineered into cord blood CD34 + cells (n= 12 recipient mice sourced from 4 separate donors for each HD, PV, and MF). (G) Representative flow cytometry plots showing intracellular pSTAT4 staining in Tet2 Δ/+ BM cells in WT or Jak2 V617F backgrounds. (H-I) Median fluorescent intensity (MFI) of intracellular pSTAT4 levels in (H) whole BM and (I) HSPCs (n = 5 / group). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 To discern the most dysregulated signaling pathways that may be driving the observed transcriptional changes, over-representation analysis (ORA) was performed on the identified DEGs. ORA identified IL-12-mediated signaling as the most significantly dysregulated cancer-related pathway between Tet2 -mutant HSPCs isolated from a Jak2 V617F -mutant background compared to from a WT background ( Fig. 4C ) . To further classify over-represented pathways, DEGs were quantitatively scored based on how frequently they appear in a cancer-related pathway relative to their frequency across all cancer-related pathways and used to weight gene set terms by their relevance across the dataset. Uniform manifold approximation and projection (UMAP) was then applied for dimensionality reduction to visualize relationships between enriched pathways. Again, among the identified clusters, IL-12-mediated signaling emerged as the most highly enriched pathway. Adjacent points in the UMAP space, inferring biological relevance of over-represented gene sets, included TNF receptor signaling and IL-12 signaling mediated by STAT4, highlighting these related signaling pathways as potential selection factors for Tet2 Δ/+ HSPC expansion in a Jak2 V617F -mutant environment (Fig. S6) . It is well-documented that MPN cells secrete high levels of many pro-inflammatory cytokines( 24 – 27 ). Moreover, we and others have shown that many common CH mutations impart growth advantages to the mutant clones under different conditions of inflammation( 28 – 32 ). To determine if the observed gene expression changes in Tet2 Δ/+ HSPCs in a Jak2 V617F -mutant environment were associated with altered cytokine levels due to the MPN cells, global cytokine profiling of PB serum from murine chimera and PDX experiments was performed. The overlap of cytokines increased in MPN models compared to relevant control comparators revealed four candidates - IL-1, IL-12, IL-27, and TNFα (Fig. S7A) . We sought to determine if any of these cytokines might create a competitive advantage for TET2 -mutant clones. CB-derived CD34 + cells were CRISPR/Cas9-engineered with TET2 mutations and cultured with each candidate cytokine at concentrations ranging from 1-100 ng/mL for 6-days. TNFα and IL-12 were the most effective in accelerating TET2 -mutant cell proliferation in vitro (Fig. S7B) . Moreover, from in vivo studies, increasing IL-12 and TNFα levels correlated with TET2 -mutant clonal expansion in murine ( Fig. 4D,E ) and PDX transplants ( Fig. 4F ) . This effect was most pronounced in PDX mice established from PV patient cells ( Fig. 4F ) . To determine the source of the aberrant signaling, IL-12 and TNFα levels were measured in serum from transplant donor mice of each genotype. IL-12 levels were significantly elevated in Jak2 V617F -mutant mice compared to WT and Tet2 Δ/+ donor mice. Conversely, TNFα was significantly elevated in Tet2 Δ/+ donor mice (Fig. S8) . Notably, TNFα has been reported to correlate with TET2 -mutant CH and IL-12 is a known TNFα stimulator that is elevated in MPN patients( 33 – 38 ). This suggests a model whereby IL-12 secreted by MPN cells induces TNFα over-production by Tet2 -mutant cells to condition an environment that fosters their development. We hypothesized that IL-12 secreted by MPN cells directly acts upon TET2 -mutant cells to phosphorylate STAT4, a key mediator of IL-12 signaling( 39 – 41 ). Intracellular flow cytometry analysis ( Fig. 4G ) revealed that pSTAT4 levels were elevated in Tet2 Δ/+ BM cells isolated from a Jak2 V617F -mutant background 10-weeks post-transplant compared to those isolated from a WT background or from WT cells from either background ( Fig. 4H ) . Strikingly, the pSTAT4 elevation was most significant in the Tet2 Δ/+ HSPC population isolated from a Jak2 V617F -mutant environment ( Fig. 4I ). This indicates a potential mechanistic link between IL-12 signaling and Tet2 Δ/+ HSPC proliferation in a Jak2 V617F -mutant context, suggesting that targeting this pathway could mitigate the competitive advantage in this genetic setting. Inhibition of Inflammatory Cytokines Mitigates the Competitive Advantage of TET2 -Mutant Cells in a Jak2 -Mutant Environment With the identification of IL-12 and TNFα as potential selective pressures for independent Tet2 -mutant clone expansion in parallel to MPN, we aimed to evaluate the functional consequences of disrupting these pathways. To enhance the clinical relevance, we replicated mutational burdens that more closely mirror those observed in MPN patients with input thresholds determined by the Jak2 V617F titration experiment. As such, chimeras utilized in functional studies were established by transplanting 1.5×10 6 Jak2 V617F BM cells (CD45.2) with 4.5×10 5 test BM (CD45.1/2) and 1.05×10 6 WT support cells (CD45.1) into lethally irradiated recipients (CD45.1) to produce a starting fraction of test BM of approximately 15% in an environment composed of approximately 50% Jak2 V617F -mutant host BM. First, to assess the implications of TNFα on the observed competitive advantage of Tet2 -mutant clones, Vav-Cre; Tet2 fl/+ mice were crossed with a TNFα-receptor genetic deletion model (p55/p75 germline knockout = “KO”) to establish mice wherein Tet2 -mutant hematopoietic cells lack receptors for TNFα. To determine the effect of ameliorating TNFα signaling in Tet2 -mutant cells, BM cells from either Jak2 V617F or WT mice were mixed with either Tet2 Δ/ + , TNFαr p55 -/- p75 -/- (= “ TNFr KO ”), Tet2 Δ/+ / TNFr KO , or WT test BM and co-transplanted with WT support. Blood ( Fig. 5A-C ) and BM analysis ( Fig. 5D ) revealed the competitive advantage of Tet2 -mutant cells in a Jak2 V617F -mutant environment was significantly blunted by genetic deletion of TNFα receptors on the Tet2 -mutant cells. Download figure Open in new tab Fig. 5. Inhibition of Inflammatory Cytokines Mitigates the Competitive Advantage of TET2 -Mutant Cells in a Jak2 -Mutant Environment. (A, B) Peripheral blood engraftment of WT control, Tet2 Δ/+ , TNFr KO and Tet2 Δ/+ ; TNFr KO , test cells in (A) WT or (B) a Jak2 V617F host BM (n= 5 / group). ( C ) 16-week peripheral blood engraftment of Tet2 Δ/+ and Tet2 Δ/+ , TNFr KO test cells in WT or Jak2 V617F host BM (n= 5 / group). ( D ) 18-week BM engraftment of Tet2 Δ/+ and Tet2 Δ/+ , TNFr KO test cells in WT or Jak2 V617F host BM (n= 5 / group). ( E-H ) Tet2 Δ/+ test cell (E) 16-week PB engraftment, (F) 18-week BM engraftment, (G) HSPC number and (H) HSC number chimeric mice receiving neutralizing antibodies against IL-12 and/or TNFα (n= 4-5 / group). (I) qPCR gene expression analysis of Tet2 Δ/+ HSPCs from indicated treatments of genetic models depicting levels of indicated genes (normalized to non-treated controls; n = 2-3 / group). (J-L) Analysis of (J) spleen weight, (K) hematocrit and (L) modified read fold change for CRISPR engineered TET2 mutations in cord blood CD34 + cells in treatment-control and IL-12-neutralizer treated PDX mice co-transplanted with PV UPN:702759 CD34 + cells (n= 2-3 / group). (M) Schematic depicting mechanism of PB cells providing a selective advantage to independent TET2 -mutant clones through cytokine support. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 To evaluate this finding in a more translational system, we administered murine biosimilars of TNFα and IL-12 monoclonal neutralizing antibodies (adalimumab / ustekinumab respectively) into mouse chimeric models between weeks 4-10 post-transplant. Inhibition of TNFα and IL-12 dramatically reduced Tet2 Δ/+ cell engraftment in a Jak2 V617F -mutant background in the PB ( Fig. 5E ) , BM ( Fig. 5F ) and HSPC populations ( Fig. 5G ) . Notably, neutralization of IL-12 and TNFα suppressed Tet2 Δ/+ cells down to the HSC level – an effect most significantly achieved by IL-12 neutralization ( Fig 5H ) . Gene expression analysis of Tet2 Δ/+ HSPCs isolated from a Jak2 V617F -mutant background at the end of the 18-week study from control and treated cohorts showed targeting the IL-12/TNFα axis normalized dysregulation in proliferation (Mki67) and self-renewal (Hoxa10, Hlf, Fgd5 , and Mecom) pathways. Similar normalization of these abnormal gene expression programs was also observed in Tet2 Δ/+ HSPCs genetically deficient for TNFα receptors ( Fig. 5I ) . Thus, genetic and pharmacological inhibition of aberrant MPN cytokine signaling abrogates the competitive advantage of Tet2 Δ/+ HSPCs in the presence of Jak2 V617F -mutant cells. To evaluate the effect of IL-12 neutralization in a human system, we utilized a JAK2 V617F -mutant PV patient sample that previously demonstrated support of parallel TET2 -mutant clones (UPN:702759). 1×10 5 CD34 + PV patient cells were co-transplanted with 2.0×10 4 CB-derived CD34 + cells harboring gRNA-mediated TET2 mutations. Between weeks 10-16 post-transplant, a human IL-12 neutralizing agent was administered to the treatment group. Control mice exhibited signs of disease progression characterized by increased spleen weight and decreased hematocrit, while mice receiving the IL-12 neutralizing agent retained PV-like pathologies ( Fig. 5J-K ) . Strikingly, IL-12 neutralization was able to mitigate the competitive advantage of TET2 -mutant clones in the presence of PV patient cells ( Fig. 5L ) . These findings reveal that targeting IL-12 could be a potential therapeutic approach for restricting TET2 -mutant CH in the background of an existing JAK2 V617F -mutant driven MPN to minimize future risk of disease progression to pAML ( Fig. 5M ) . Conclusion These data establish that JAK2 V617F -mutant cells potentiate parallel expansion of independent clones as a non-classical trajectory of disease progression in MPN. We show that MPN cells drive this parallel evolution through an IL-12/TNFα cytokine axis, which biases Tet2 -mutant progenitors towards increased proliferation and myeloid differentiation. Genetic and pharmacological inhibition of IL-12 and TNFα resulted in both functional and molecular rescue of these phenotypes. Our findings provide crucial insights into how sAML which lacks the JAK2 V617F driver mutation can evolve from an antecedent JAK2 V617F -mutant MPN. These results highlight a specific example of clonal dynamics and cell competition within a JAK2 V617F -mutant context; however, it is important to acknowledge that clones with other CH mutations (e.g. DNMT3A) as well as MPNs driven by other mutations (CALR, MPL) may confer different patterns of clonal evolution. We aim to leverage these findings to enhance disease surveillance in MPN populations. With improved genomic monitoring, targeted interventional therapies to prevent the expansion of emerging pre-leukemic clones could reduce risk of parallel disease evolution in MPN, a significant therapeutic advantage for a substantial subset of patients. Funding National Institutes of Health grant HL147978 (GAC) National Institutes of Health grant CA236819 (GAC) National Institutes of Health grant DK124883 (GAC) Edwards P. Evans Foundation (GAC) Blood Cancer United grant 6667-23 (GAC) American Cancer Society grant CSCC-RSG-23-991417-01-CSCC (GAC) National Institutes of Health grant K99CA296777 (TMP) National Institutes of Health grant T32HL007088 (ALY) National Institutes of Health grant DP50D039424 (ALY) National Institutes of Health grant R01HL134952 (STO) Siteman Cancer Center Team Science award (GAC, STO) Author Contributions Conceptualization: TMP, STO, GAC Methodology: TMP, AK, IXR, ALY, GAC Investigation: TMP, AK, IXR, ALY, DO, JA, MC, GAC Visualization: TMP, IXR, ALY Funding acquisition: GAC Project administration: TMP, JA, GAC Supervision: GAC Writing – original draft: TMP, GAC Writing – review and editing: TMP, GAC Competing interests The authors declare the following competing interests (unrelated to this work): TMP has advisory role positions with the MPN Research Foundation and has performed consulting for PharmaEssentia and Silence Therapeutics. ALY has performed consulting for BioGenerator and is a co-founder, CEO, and shareholder of Pairidex Inc. STO has served as a consultant for Abbvie, Bristol Myers Squibb, Cogent, Constellation/Morphosys, CTI BioPharma/Sobi, Geron, Incyte, Morphic, Protagonist, and Sierra Oncology/GSK. GAC has performed consulting and received research funding from Incyte, Ajax Therapeutics, ReNAgade Therapeutics Management, and Atavistik Bio and is a co-founder, member of the scientific advisory board, and shareholder of Pairidex Inc. Data and materials availability Data not available in the main text or the supplementary materials are available through request to the corresponding author. All raw read data (FASTQ files) for RNA sequencing are publicly available at the Gene Expression Omnibus database accession number GSE308233. Supplementary Materials Materials and Methods Figs. S1 to S8 Table S1 to S2 References ( 42 - 50 ) Data S1 Acknowledgments We thank all members of the Challen laboratory. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO. for the use of the Siteman Flow Cytometry Core facilities supported in part by an NCI Cancer Center Support Grant #P30CA091842. We thank Dr. Jorge Di Paola for generously providing TNFα-receptor germline knock-out mice. Tp53 R172H mice were originally developed by Dr. Guillermina Lozano (MD Anderson Cancer Center). This publication is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. T.M.P is a Fellow of Blood Cancer United. A.K. was supported by the American Society of Hematology (ASH) Graduate Hematology Award. A.L.Y. was supported by the ASH Research Training Award for Fellows and Scholar Award, and the Edward P. Evans Foundation Young Investigator Award. I.X.R. was supported by NIH P30 CA091842. Funder Information Declared Leukemia and Lymphoma Society, https://ror.org/04m0xav37 , 6667-23 American Society of Hematology, https://ror.org/02nw48b86 Edward P. Evans Foundation, https://ror.org/03h22gm35 National Institutes of Health, https://ror.org/01cwqze88 , HL147978 , CA236819 , DK124883 , K99CA296777 , R01HL134952 , DP50D039424 American Cancer Society , CSCC-RSG-23-991417-01-CSCC References and Notes 1. ↵ R. Kralovics , F. Passamonti , A. S. Buser , S.-S. Teo , R. Tiedt , J. R. Passweg , A. Tichelli , M. Cazzola , R. C. Skoda , A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders . N Engl J Med 352 , 1779 – 1790 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 2. E. J. Baxter , L. M. Scott , P. J. Campbell , C. East , N. Fourouclas , S. Swanton , G. S. Vassiliou , A. J. Bench , E. M. Boyd , N. Curtin , M. A. Scott , W. N. Erber , A. R. Green , Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders . 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