Wild-Type p53 Overexpression in NPM1-Mutated AML: Potential Implications for Disease Biology and Therapy Response

Wild-Type p53 Overexpression in NPM1-Mutated AML: Potential Implications for Disease Biology and Therapy Response | 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 Wild-Type p53 Overexpression in NPM1 -Mutated AML: Potential Implications for Disease Biology and Therapy Response Paul Barone, Siarhei Dzedzik, Ashley Kleinman, Christopher Chin, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5214655/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Somatic mutations in nucleophosmin ( NPM1 ) are key in defining a common subtype of acute myeloid leukemia ( NPM1 -AML), characterized by chromosomal stability and favorable therapeutic responses. However, some patients exhibit a suboptimal response to initial treatment, and relapses are common, highlighting the need for novel biomarkers. Notably, NPM1 and TP53 mutations rarely co-occur in AML, where TP53 mutations correlate with aneuploidy and poor survival outcomes. Here, we present transcriptomic and proteomic evidence of unexpectedly high wild-type (WT) TP53 /p53 expression in a subset of NPM1 -AMLs at baseline. Analyses of the Beat AML cohort indicate that NPM1 -AMLs generally express more TP53 than NPM1 / TP53 -WT AMLs. Among seventy-four NPM1 -AML samples, those with the lowest TP53 co-expression are enriched for downregulated signatures linked to DNA repair, apoptosis, and the cell cycle. By profiling thirty-three primary patient samples using multiplex immunofluorescence staining with single cell-based quantitative digital image analysis, we observed increased p53 expression in NPM1 -mutant cells compared to WT cells. Importantly, patients with primary refractory disease showed low p53 co-expression at diagnosis. These findings suggest that WT-p53 might play a crucial role in the biological and clinical characteristics seen in NPM1 -AML. Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia Health sciences/Pathogenesis/Oncogenesis Health sciences/Medical research/Translational research Biological sciences/Cell biology/Cell death/Apoptosis NPM1 AML p53 biomarker Figures Figure 1 Figure 2 INTRODUCTION In the latest World Health Organization (WHO) and International Consensus Classification (ICC) diagnostic schema for hematolymphoid neoplasms acute myeloid leukemias (AML) are now largely genetically-defined. 1 , 2 At seemingly opposite ends of the clinicopathologic and genomic spectra lie AML with mutated NPM1 ( NPM1 -AML) and AML (and precursor states) harboring TP53 abnormalities ( TP53 -AML). Approximately 30% of de novo AML cases are defined by mutations in NPM1 , and are commonly associated with a normal karyotype, response to induction therapy, and a relatively favorable clinical course. In contrast, TP53 -AMLs are characterized by aneuploidy, uniformly poor response to standard-of-care therapeutic strategies, and a dismal prognosis. The function of wild-type NPM1 is multifaceted, with roles in ribosome biogenesis, genetic stability, cell proliferation, and apoptosis. 3 The latter two of these processes are executed through stabilization of p53, a critical cell cycle regulator and pro-apoptotic protein. This stabilization is mediated, in part, through MDM2, a p53-E3 ubiquitin ligase. This ligase is normally active in the nucleoplasm; however, in response to stress and nucleolar damage, WT-NPM1 disengages from the nucleolus and binds Hdm2/Mdm2, thereby inactivating it and allowing p53 to avoid degradation. WT-NPM1 and WT-p53 are therefore known to be functionally intertwined. Sequencing studies involving large AML cohorts have revealed NPM1 and TP53 mutations to be essentially mutually exclusive. 4 – 6 In our diagnostic hematopathology practice, we commonly utilize antibody-based immunohistochemistry for mutant NPM1 and p53 proteins as surrogate markers for their underlying genetic lesions as part of the initial examination of AML bone marrow biopsy tissues. 7 – 10 While moderate to strong p53 staining in a significant proportion of tumor cells (i.e. aberrant accumulation of mutant protein) is highly suggestive of a missense mutation in the TP53 DNA binding domain, we encountered a similar pattern in several NPM1 -AMLs confirmed to be TP53 -wild type by sequencing studies. Therefore, we sought to explore the frequency of this protein expression pattern, to identify possible mechanisms for wild-type p53 overexpression in NPM1 -AML, and to assess for wild-type p53 pathway activity in this relatively common AML subtype. METHODS Re-Analysis of RNA Sequencing Data Publicly available RNA sequencing data from the Beat AML cohort 6 was accessed through cBioPortal. Cases were filtered to include initial diagnosis only, and limma (RRID:SCR_010943) was used on CPM values for differential expression, and to regress differences in sample site and response. 11 GSEA analysis was performed on the regressed log2FC data using MSigDB signatures. 12 Analysis code is available upon request. Re-Analysis of Chromatin Immunoprecipitation Sequencing (ChIP-SEQ) Data ChIP bigwig files with and without degron treatment were downloaded from GSE197387. The TP53 locus was visualized by IGV, but there was no discernable difference based on NPM1 degron treatment. Case Selection (for Multiplex Immunofluorescence Tissue Staining) Archival (paraffin-embedded) bone marrow aspirate specimens obtained from the posterior iliac crest were used in the study. Specimens were originally collected for routine diagnostic evaluation of suspected leukemia at Weill Cornell Medical College/NewYork-Presbyterian Hospital (WCM/NYP). A total of 45 individuals were included, including patients with NPM1 -mutated AML (n = 33) [ Table 1 ], TP53 -mutated AML (n = 5), and NPM1 / TP53 -wild-type AML with normal karyotype (n = 7). Paraffin-embedded tissue specimens were selected and de-identified prior to testing, upon approval by the institutional review board (IRB). Clinical and laboratory data were also collected. Cytogenetic and next-generation sequencing data for all cases were generated and collected as previously described; 9 all cases were assessed for NPM1 and TP53 coding sequence mutations. Chromogenic Immunohistochemistry and Multiplex Immunofluorescence (MxIF) Tissue Staining Multiplexed immunofluorescence (MxIF) was performed using the Opal system (Akoya Biosciences, Marlborough, MA) by staining 4 micron-thick formalin-fixed, paraffin-embedded aspirate fluid (“clot sections”) in a Bond RX automated tissue stainer (Leica Biosystems, Buffalo Grove, IL), as described previously. 13 Antibodies were selected from a menu of extensively validated and clinically tested clones in our CLIA laboratory (Weill Cornell Medicine/NewYork-Presbyterian Hospital, New York, NY) [ Supplemental Methods ]. MxIF Imaging and Digital Image Analysis Whole slide MxIF images were captured using the Phenoimager platform (Akoya Biosciences); following visual QC in Phenochart (Akoya), images were analyzed in HALO (v3.6.4134.95, Indica Labs, Albuquerque, NM) by two hematopathologists (P.B., S.S.P.), including cell segmentation, visual thresholding of antigen expression for individual markers, and phenotyping (e.g., NPM1 mutant vs. WT, p53 positive vs. negative). Single cell matrices were output and further analyzed in R v.4.4.1 (RStudio version 2023.06.1 Build 524). Statistical Analyses Statistical analyses were conducted in R v.4.4.1 (RStudio version 2023.06.1 Build 524). Wilcoxon signed rank exact test was used to compare the proportions of positive p53 expression and mean fluorescence intensity in NPM1 mutant versus wild type cells, as well as the proportions of p53 expression in NPM1 -mutated cells between complete remission with or without complete count recovery (CR/CRi) and persistent disease (PD) groups. A p-value of less than 0.05 was considered statistically significant. The same p-value was used to assess differential gene expression; no adjustment was performed as only TP53 values were assessed. RESULTS Wild Type-TP53 is more highly expressed in NPM1-mutated AML compared to other AML subtypes In our clinical diagnostic hematopathology practice we routinely perform screening for TP53 and NPM1 mutations by chromogenic immunohistochemistry (IHC) as a component of new AML evaluations. 8,10 In select cases of molecularly- confirmed NPM1 -mutated/ TP53 -WT AML, we have observed p53 expression at a level often observed in the cases harboring missense mutations in the DNA binding domain of TP53 ( Figure 1A, representative case ). We hypothesized that the elevated expression of WT-p53 could be a result of increased TP53 gene expression in at least a subset of NPM1 -AML cases. Based on an analysis of the BeatAML cohort dataset, 6 we found that NPM1 -AML cases (n=74) are associated with significantly higher TP53 expression than TP53 -WT/ NPM1 -WT cases (n=190) [p= 0.047, multivariate limma model] ( Figure 1B ). We considered the possibility that this difference may be driven by mutations with prognostic significance co-occurring with NPM1 ( FLT3 -ITD, FLT3 -TKD, DNMT3A , SF3B1 , SRSF2 , U2AF1 ); however, exclusion of these genes by comparing cases of NPM1 -AML to NPM1 -WT/ TP53 -WT/gene mutation-positive cases revealed a similar result [p=0.018, multivariate limma model] ( Figure 1C ). Furthermore, we observed no effect of specimen type (e.g., peripheral blood, bone marrow, or leukapheresis product) on TP53 expression level ( Figure 1D ). p53-associated gene modules are upregulated in NPM1 - mutated AML and correlate with wild-type TP53 expression level To assess the activity of WT-p53 signaling we focused on known p53-associated gene sets. By comparing NPM1 -AML with TP53 -AML cases (n=17), we first established the pattern of up-/down-regulation for gene sets associated with DNA repair, apoptosis, and cell cycle pathways as a function of TP53 mutation ( Figure 1E ). Despite the genetic heterogeneity among NPM1 -WT cases, relative to NPM1 -AML cases the -WT group exhibited a similar gene set enrichment pattern as seen for TP53 -AML cases ( Figure 1F) . We next performed a similar analysis restricted only to NPM1 -AML cases, comparing the uppermost (n=19) and lowermost (n=19) quartiles for TP53 gene expression; we noted a near-complete overlap in the pattern of up- and/or downregulation across DNA repair, apoptosis, and cell cycle gene sets as we observed when comparing NPM1 -AML and TP53 -AML cases ( Figure 1G ), suggesting that the activity of these pathways may be directly influenced by WT-p53 dosage within the context of NPM1 -AML. WT-p53 protein is over-expressed in NPM1-mutated cells in AML and correlates with remission status post-induction therapy Based on our initial findings of increased WT-p53 protein expression in NPM1 -mutated cells in a case of AML, and evidence of increased TP53 expression in NPM1 -AML cases within the BeatAML cohort, we further assessed p53 protein expression in a cohort of primary human bone marrow tissue samples from our institution ( Table 1 ). Using a multiplex immunofluorescence-based phenotyping assay coupled with digital image analysis, we phenotyped cells in situ at single cell resolution, identifying them as NPM1 -mutant or -WT using a mutant protein-specific antibody, and then evaluated the frequency and intensity of p53 expression ( Figure 2A ). The median number of nucleated cells analyzed per case was 8,957 (range: 1,869 - 31,151) [ Supplemental Figure 1 ]. Within each case, a greater proportion of NPM1 -mutated cells were p53-positive, compared to NPM1 -WT cells [p10% of NPM1 ­-mutant cells to be p53-positive in 24 of 33 cases (73%). Similarly, the mean fluorescence intensity (MFI) of p53 was higher in NPM1 -mutant compared to WT cells [p<0.001, paired Wilcoxon test] ( Figure 2D-E ). We observed no significant difference in p53 proportion between patients above or below the age of 60, in those presenting with or without leukocytosis, or with respect to peripheral blood or bone marrow blast percentage (data not shown). We considered the possibility that p53 expression may simply be associated with a non-G0 state of the leukemic cells; however, we observed no correlation between p53 and Ki67 expression ( Supplemental Figure 2 ). We also wondered if p53 overexpression could be a result of diminished MDM2-mediated degradation due to cytoplasmic sequestration of MDM2 by mutant NPM1; however, an analysis of the few highest p53 co-expressors revealed no significant cytoplasmic MDM2 signal by multiplex immunofluorescence ( Supplemental Figure 3 ). Furthermore, we found no significant difference in p53 co-expression frequency based on presence or absence of common co-mutations (e.g. FLT3 -ITD, DNMT3A , IDH1/2 ) [ Supplemental Figure 4 ]. As a proportion of total nucleated cells, p53 was most frequently detected in TP53 -AML cases, as anticipated; NPM1 -AML cases included a range of p53 expression frequency, with a subset exhibiting p53 expression near the level found in TP53 -AML cases. We observed no significant difference in p53 co-expression among total nucleated cells between NPM1 -AML and a small comparison group of normal karyotype NPM1 -WT cases (p>0.05) [ Supplemental Figure 5 ]. Similarly, p53 MFI was significantly higher in TP53 -mutated versus all TP53 -WT cases ( Supplemental Figure 6 ). Post-induction remission status was available for 27 of the 33 cases analyzed. Interestingly, we found a significantly higher p53-positive proportion among NPM1 -mutated cells at diagnosis in patients who achieved complete remission with or without complete count recovery (CR/CRi, n=21) compared to patients with grossly persistent disease (PD, n=6) [median 0.219 vs. 0.086, p=0.018] [ Figure 2F ]. Of note, we found no difference in the frequency of FLT3 -ITD co-mutations in CR/CRi versus PD patients (p>0.05). DISCUSSION The basis for many of the biological (e.g. normal karyotype/genomic stability) and clinical (e.g. favorable response to induction therapy) features of NPM1 -AML remains largely unknown. While many patients have a favorable clinical course, a subset experience recurrent disease after a period of remission, or more rarely, refractory disease after initial therapy. Many, but not all, patients with this trajectory can be anticipated using well-recognized adverse clinical and/or genetic features, although other biomarkers with predictive potential remain to be identified. In stark contrast, TP53 -AML is associated with uniformly dismal outcomes; and interestingly, NPM1 and TP53 mutations are considered essentially mutually exclusive. 4 – 6 Through routine use of protein immunohistochemistry to assess p53 and mutant NPM1 protein expression status in the diagnostic evaluation of AML cases, we observed cases of NPM1 -AML with a high frequency and intensity of WT-p53 expression, leading us to hypothesize that the common clinical and biological features of NPM1 -AML could be explained, at least in part, by retained WT-p53 activity. An exploration of publicly available RNA sequencing data from the Beat AML cohort revealed higher TP53 gene expression in NPM1 -AML cases compared to NPM1 -WT AML cases. Of note, we observed no difference in TP53 expression level among NPM1 -AML cases upon separating them based on mutational status of genes with known prognostic significance (e.g. FLT3 -ITD, DNMT3A ). Given recently published data demonstrating that mutant NPM1 protein directly binds to chromatin to modify gene expression, 14 , 15 we explored the possibility that mutant NPM1 could be directly driving TP53 expression, but did not find compelling evidence to support this mechanism. We then used gene set enrichment analysis to evaluate the functional status of p53, focusing primarily on pathways related to DNA repair, apoptosis, and cell cycle regulation. We observed an overlapping pattern of positively or negatively enriched gene sets for NPM1 -AML cases when compared to either TP53 -AML or NPM1 -WT cases. Interestingly, separating NPM1 -AML cases into those with high and low TP53 co-expression recapitulated the gene set enrichment differences observed between NPM1 -AML and TP53 -AML cases; this result suggests that low TP53 co-expression in NPM1 -AML may approximate the absence of a normally functioning p53 protein. We finally assessed p53 protein expression in a cohort of patient samples from our institution using a multiplex immunofluorescence assay with digital image analysis, capitalizing on the concurrent application of a mutant NPM1 protein-specific antibody to define the mutational status of individual cells. These studies revealed elevated p53 expression in > 10% of NPM1 -mutant cells in 73% of the analyzed cases, with a higher frequency and intensity of p53 expression in NPM1 mutant relative to WT cells observed in all cases. Interestingly, patients with grossly persistent disease following induction therapy were characterized by a significantly lower frequency of p53 expression at diagnosis than those who achieved complete remission. Our study is limited by its retrospective design, the small internal cohort of patient samples studied, and largely correlative findings. However, to the best of our knowledge, our data provide the first evidence that many NPM1 -AML cases are characterized by elevated TP53 gene and p53 protein expression at baseline. It remains possible that this profile could be driven by either 1) MDM2 sequestration by residual WT-NPM1 protein with consequently reduced MDM2-mediated p53 degradation, or 2) p53 stabilization via phosphorylation at select serine residues. 16 Although mechanistic studies will be required to explore these and other potential mechanisms underlying increased p53 expression, we present evidence suggesting a role for increased TP53 gene expression; this finding is associated with activity of p53-mediated gene expression pathways linked to DNA repair, apoptosis, and cell cycle regulation opposite that observed in TP53 -AML. We therefore hypothesize that, while increased p53 activity might contribute to the intrinsic biological and clinical features of most NPM1 -AML cases, low p53 expression in a subset of cases at diagnosis could represent a potential biomarker of unfavorable disease worthy of further exploration in larger cohorts. Declarations AUTHOR CONTRIBUTIONS Experiments were designed, executed and/or analyzed by P.B., I.V., A.S.K., C.R.C., C.M., M.G., J.K., J.A.F., C.E.M., and S.S.P. I.V. performed multiplex tissue staining and imaging. J.K., J.A.F., G.J.R. and P.D. reviewed clinical data. The manuscript was written by P.B. and S.S.P. with input from all authors. P.B. and S.S.P. conceived of and designed the study. S.S.P. supervised the work. ACKNOWLEDGEMENTS This work was supported by the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College (start-up funding to S.S.P.). C.R.C. is supported by an NIH post-doctoral training grant (T32AR071302). Tissue staining and imaging was performed in the Multiparametric In Situ Imaging (MISI) Laboratory of the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College. DATA AVAILABILITY Analysis code and data are available upon reasonable written request to the corresponding author (e-mail: [email protected] ). 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Pasqualucci L, Liso A, Martelli MP, et al. Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: Impact on WHO classification. Blood. 2006;108(13):4146–4155. Patel SS, Pinkus GS, Ritterhouse LL, et al. High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia. Am. J. Hematol. 2019;94(8):921–928. Lopez A, Patel S, Geyer JT, et al. Comparison of Multiple Clinical Testing Modalities for Assessment of NPM1-Mutant AML. Front. Oncol. 2021;11:701318. Tashakori M, Kadia T, Loghavi S, et al. TP53 copy number and protein expression inform mutation status across risk categories in acute myeloid leukemia. Blood. 2022;140(1):58–72. Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U. S. A. 2005;102(43):15545–15550. Sarachakov A, Varlamova A, Svelolkin V, et al. Spatial Mapping of Human Hematopoiesis at Single Cell Resolution Reveals Aging-Associated Topographic Remodeling. Blood . 2023;blood.2023021280. Uckelmann HJ, Haarer EL, Takeda R, et al. Mutant NPM1 directly regulates oncogenic transcription in acute myeloid leukemia. Cancer Discov. 2022;CD-22-0366. Wang XQD, Fan D, Han Q, et al. Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia. Cancer Discov. 2023;13(3):724–745. Ashcroft M, Kubbutat MH, Vousden KH. Regulation of p53 function and stability by phosphorylation. Mol. Cell. Biol. 1999;19(3):1751–1758. Additional Declarations There is NO conflict of interest to disclose. Table 1 is available in the supplementary files section. Supplementary Files NPM1p53ManuscriptSuppMethodsLeukemia090324.docx Supplemental Methods NPM1p53SupplementalData082124.pdf Supplemental Data NPM1p53Table1082124.pdf Table 1 Cite Share Download PDF Status: Under Review Version 1 posted Unknown event 04 Dec, 2024 Editorial decision: Reject after peer review 03 Dec, 2024 Review # 2 received at journal 05 Nov, 2024 Reviewer # 2 agreed at journal 23 Oct, 2024 Reviewer # 1 agreed at journal 20 Oct, 2024 Reviewers invited by journal 08 Oct, 2024 Editor assigned by journal 08 Oct, 2024 Submission checks completed at journal 08 Oct, 2024 First submitted to journal 08 Oct, 2024 Unknown event 07 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5214655","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":363657988,"identity":"8180272e-ca6f-4128-838a-7e6e48fe88ad","order_by":0,"name":"Paul Barone","email":"","orcid":"","institution":"Weill Cornell Medicine","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Barone","suffix":""},{"id":363657989,"identity":"f785b9bd-d472-4dcf-9930-195c4f510469","order_by":1,"name":"Siarhei Dzedzik","email":"","orcid":"","institution":"Weill Cornell 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23:00:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5214655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5214655/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66285471,"identity":"8a41c754-5c31-4a05-9759-5bfcdc8b263b","added_by":"auto","created_at":"2024-10-09 16:56:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":915714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is over-expressed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNPM1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-AML relative to other AMLs and associated with p53 pathway activity.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eRepresentative case of \u003cem\u003eNPM1\u003c/em\u003e-AML. Bone marrow aspirate smear preparation shows a predominance of blasts with monocytic features (top left, Wright-Giemsa, 1000x). Histologic evaluation of the trephine biopsy shows sheets of medium to large sized blasts with monocytic features (top right, hematoxylin and eosin, 600x). Duplex chromogenic immunohistochemistry for p53 (brown) and mutant NPM1 (red) proteins reveals frequent moderate to strong p53 expression in mutant NPM1-positive cells (bottom, p53 (DAB)/mutant NPM1 (Fast Red), 600x). \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eTP53\u003c/em\u003e is more highly expressed in \u003cem\u003eNPM1\u003c/em\u003e-AML compared to \u003cem\u003eNPM1-\u003c/em\u003eWT AML cases (p= 0.047, multivariate limma model). \u003cstrong\u003eC. \u003c/strong\u003eAmong \u003cem\u003eNPM1\u003c/em\u003e-AML cases, \u003cem\u003eTP53\u003c/em\u003e is more highly expressed in those lacking co-mutation in \u003cem\u003eDNMT3A\u003c/em\u003e, \u003cem\u003eFLT3\u003c/em\u003e, \u003cem\u003eSF3B1\u003c/em\u003e, \u003cem\u003eSRSF2\u003c/em\u003e, \u003cem\u003eU2AF1\u003c/em\u003e (p= 0.018, multivariate limma model). \u003cstrong\u003eD. \u003c/strong\u003e\u003cem\u003eTP53\u003c/em\u003e is more highly expressed in \u003cem\u003eNPM1\u003c/em\u003e-AML compared to \u003cem\u003eNPM1\u003c/em\u003e-WT AML, irrespective of sample type; statistical analysis was performed as part of panel \u003cstrong\u003eB \u003c/strong\u003edata. \u003cstrong\u003eE-G\u003c/strong\u003e. Gene set enrichment analyses (GSEA) of msigdb pathways colored by normalized enrichment score (NES), with significance (p-adj \u0026lt; 0.05) denoted by an asterisk. p53-mediated pathways related to DNA repair, apoptosis, and cell cycle are more active in \u003cem\u003eNPM1\u003c/em\u003e-AML relative to \u003cem\u003eTP53-\u003c/em\u003eAML (\u003cstrong\u003eE\u003c/strong\u003e), and several are more active in \u003cem\u003eNPM1\u003c/em\u003e-AML compared to \u003cem\u003eNPM1\u003c/em\u003e-WT/\u003cem\u003eTP53-\u003c/em\u003eWT AML (\u003cstrong\u003eF\u003c/strong\u003e). Among \u003cem\u003eNPM1\u003c/em\u003e-AMLs, relative to cases in the uppermost quartile for \u003cem\u003eTP53\u003c/em\u003e co-expression, those in the lowermost quartile exhibit pathway enrichment similar to that observed for \u003cem\u003eTP53-\u003c/em\u003emutated cases (\u003cstrong\u003eG\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"20240903Fig1NPM1AMLsigaskalt.png","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/ea5920cb489db5fe03bde6bb.png"},{"id":66284962,"identity":"92a2bf6a-8145-4f9a-bbc7-a25b8496b8c0","added_by":"auto","created_at":"2024-10-09 16:48:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":624560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep53 is over-expressed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNPM1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-mutated cells in primary patient samples and low co-expression correlates with persistent disease post-induction therapy. A. \u003c/strong\u003eRepresentative multiplex immunofluorescence (MxIF) images from a case of \u003cem\u003eNPM1\u003c/em\u003e-AML labeled with visualization of nuclei (DAPI), mutant NPM1 [NPM1c] (green), p53 (red), and Ki67 (cyan). Mutant NPM1-positive cells exhibit frequent nuclear co-expression of p53. Frequent p53 and Ki67 co-expression is not observed (see \u003cstrong\u003eSupplemental Figure 2\u003c/strong\u003e). \u003cstrong\u003eB-C. \u003c/strong\u003eWithin each case of \u003cem\u003eNPM1\u003c/em\u003e-AML analyzed by MxIF, a greater proportion of \u003cem\u003eNPM1\u003c/em\u003e-mutated cells are p53-positive, compared to \u003cem\u003eNPM1\u003c/em\u003e-WT cells (p\u0026lt;0.001, paired Wilcoxon test). \u003cstrong\u003eD-E. \u003c/strong\u003eWithin each case of \u003cem\u003eNPM1\u003c/em\u003e-AML analyzed by MxIF, the mean fluorescence intensity (MFI) of p53 is greater in \u003cem\u003eNPM1\u003c/em\u003e-mutated cells compared to \u003cem\u003eNPM1\u003c/em\u003e-WT cells (p\u0026lt;0.001, paired Wilcoxon test). \u003cstrong\u003eF. \u003c/strong\u003eHigher p53-positive proportion among \u003cem\u003eNPM1\u003c/em\u003e-mutated cells at diagnosis in patients who achieved complete remission with or without complete count recovery (CR/CRi, n=21) post-induction compared to patients with persistent disease (PD, n=6) [median 0.219 vs. 0.086, p=0.018].\u003c/p\u003e","description":"","filename":"20240903Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/3528eacc9fd7fa081c2abb90.png"},{"id":70453489,"identity":"8aeb1b47-a12f-4399-8bb6-9727508dd667","added_by":"auto","created_at":"2024-12-03 10:08:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2379226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/2682d8cf-32af-4ce1-83df-6eed8ad5f390.pdf"},{"id":66284964,"identity":"be2055b9-b8e5-4350-b3f0-94e541e115b1","added_by":"auto","created_at":"2024-10-09 16:48:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16137,"visible":true,"origin":"","legend":"Supplemental Methods","description":"","filename":"NPM1p53ManuscriptSuppMethodsLeukemia090324.docx","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/4a6cf5d1a96c921427c8fffa.docx"},{"id":66284965,"identity":"a428048a-8b6d-48db-9ea6-3fc7250b3950","added_by":"auto","created_at":"2024-10-09 16:48:38","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":602442,"visible":true,"origin":"","legend":"Supplemental Data","description":"","filename":"NPM1p53SupplementalData082124.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/7443d0ab4605cef45503b0aa.pdf"},{"id":66284966,"identity":"987ea94e-ad1a-495c-9537-ce9b8bfc141c","added_by":"auto","created_at":"2024-10-09 16:48:38","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":136972,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"NPM1p53Table1082124.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5214655/v1/8b590c1302ccd1e9f9740306.pdf"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e conflict of interest to disclose.\u003c/p\u003e\n\u003cp\u003eTable 1 is available in the supplementary files section.\u003c/p\u003e","formattedTitle":"\u003cp\u003eWild-Type p53 Overexpression in \u003cem\u003eNPM1\u003c/em\u003e-Mutated AML: Potential Implications for Disease Biology and Therapy Response\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn the latest World Health Organization (WHO) and International Consensus Classification (ICC) diagnostic schema for hematolymphoid neoplasms acute myeloid leukemias (AML) are now largely genetically-defined.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e At seemingly opposite ends of the clinicopathologic and genomic spectra lie AML with mutated \u003cem\u003eNPM1\u003c/em\u003e (\u003cem\u003eNPM1\u003c/em\u003e-AML) and AML (and precursor states) harboring \u003cem\u003eTP53\u003c/em\u003e abnormalities (\u003cem\u003eTP53\u003c/em\u003e-AML). Approximately 30% of de novo AML cases are defined by mutations in \u003cem\u003eNPM1\u003c/em\u003e, and are commonly associated with a normal karyotype, response to induction therapy, and a relatively favorable clinical course. In contrast, \u003cem\u003eTP53\u003c/em\u003e-AMLs are characterized by aneuploidy, uniformly poor response to standard-of-care therapeutic strategies, and a dismal prognosis.\u003c/p\u003e \u003cp\u003eThe function of wild-type NPM1 is multifaceted, with roles in ribosome biogenesis, genetic stability, cell proliferation, and apoptosis.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The latter two of these processes are executed through stabilization of p53, a critical cell cycle regulator and pro-apoptotic protein. This stabilization is mediated, in part, through MDM2, a p53-E3 ubiquitin ligase. This ligase is normally active in the nucleoplasm; however, in response to stress and nucleolar damage, WT-NPM1 disengages from the nucleolus and binds Hdm2/Mdm2, thereby inactivating it and allowing p53 to avoid degradation. WT-NPM1 and WT-p53 are therefore known to be functionally intertwined.\u003c/p\u003e \u003cp\u003eSequencing studies involving large AML cohorts have revealed \u003cem\u003eNPM1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e mutations to be essentially mutually exclusive.\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e In our diagnostic hematopathology practice, we commonly utilize antibody-based immunohistochemistry for mutant NPM1 and p53 proteins as surrogate markers for their underlying genetic lesions as part of the initial examination of AML bone marrow biopsy tissues.\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e While moderate to strong p53 staining in a significant proportion of tumor cells (i.e. aberrant accumulation of mutant protein) is highly suggestive of a missense mutation in the \u003cem\u003eTP53\u003c/em\u003e DNA binding domain, we encountered a similar pattern in several \u003cem\u003eNPM1\u003c/em\u003e-AMLs confirmed to be \u003cem\u003eTP53\u003c/em\u003e-wild type by sequencing studies. Therefore, we sought to explore the frequency of this protein expression pattern, to identify possible mechanisms for wild-type p53 overexpression in \u003cem\u003eNPM1\u003c/em\u003e-AML, and to assess for wild-type p53 pathway activity in this relatively common AML subtype.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRe-Analysis of RNA Sequencing Data\u003c/h2\u003e \u003cp\u003ePublicly available RNA sequencing data from the Beat AML cohort\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e was accessed through cBioPortal. Cases were filtered to include initial diagnosis only, and limma (RRID:SCR_010943) was used on CPM values for differential expression, and to regress differences in sample site and response.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e GSEA analysis was performed on the regressed log2FC data using MSigDB signatures.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Analysis code is available upon request.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRe-Analysis of Chromatin Immunoprecipitation Sequencing (ChIP-SEQ) Data\u003c/h3\u003e\n\u003cp\u003eChIP bigwig files with and without degron treatment were downloaded from GSE197387. The \u003cem\u003eTP53\u003c/em\u003e locus was visualized by IGV, but there was no discernable difference based on \u003cem\u003eNPM1\u003c/em\u003e degron treatment.\u003c/p\u003e\n\u003ch3\u003eCase Selection (for Multiplex Immunofluorescence Tissue Staining)\u003c/h3\u003e\n\u003cp\u003eArchival (paraffin-embedded) bone marrow aspirate specimens obtained from the posterior iliac crest were used in the study. Specimens were originally collected for routine diagnostic evaluation of suspected leukemia at Weill Cornell Medical College/NewYork-Presbyterian Hospital (WCM/NYP). A total of 45 individuals were included, including patients with \u003cem\u003eNPM1\u003c/em\u003e-mutated AML (n\u0026thinsp;=\u0026thinsp;33) [\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e], \u003cem\u003eTP53\u003c/em\u003e-mutated AML (n\u0026thinsp;=\u0026thinsp;5), and \u003cem\u003eNPM1\u003c/em\u003e/\u003cem\u003eTP53\u003c/em\u003e-wild-type AML with normal karyotype (n\u0026thinsp;=\u0026thinsp;7). Paraffin-embedded tissue specimens were selected and de-identified prior to testing, upon approval by the institutional review board (IRB). Clinical and laboratory data were also collected. Cytogenetic and next-generation sequencing data for all cases were generated and collected as previously described;\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e all cases were assessed for \u003cem\u003eNPM1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e coding sequence mutations.\u003c/p\u003e\n\u003ch3\u003eChromogenic Immunohistochemistry and Multiplex Immunofluorescence (MxIF) Tissue Staining\u003c/h3\u003e\n\u003cp\u003eMultiplexed immunofluorescence (MxIF) was performed using the Opal system (Akoya Biosciences, Marlborough, MA) by staining 4 micron-thick formalin-fixed, paraffin-embedded aspirate fluid (\u0026ldquo;clot sections\u0026rdquo;) in a Bond RX automated tissue stainer (Leica Biosystems, Buffalo Grove, IL), as described previously.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Antibodies were selected from a menu of extensively validated and clinically tested clones in our CLIA laboratory (Weill Cornell Medicine/NewYork-Presbyterian Hospital, New York, NY) [\u003cb\u003eSupplemental Methods\u003c/b\u003e].\u003c/p\u003e\n\u003ch3\u003eMxIF Imaging and Digital Image Analysis\u003c/h3\u003e\n\u003cp\u003eWhole slide MxIF images were captured using the Phenoimager platform (Akoya Biosciences); following visual QC in Phenochart (Akoya), images were analyzed in HALO (v3.6.4134.95, Indica Labs, Albuquerque, NM) by two hematopathologists (P.B., S.S.P.), including cell segmentation, visual thresholding of antigen expression for individual markers, and phenotyping (e.g., NPM1 mutant vs. WT, p53 positive vs. negative). Single cell matrices were output and further analyzed in R v.4.4.1 (RStudio version 2023.06.1 Build 524).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted in R v.4.4.1 (RStudio version 2023.06.1 Build 524). Wilcoxon signed rank exact test was used to compare the proportions of positive p53 expression and mean fluorescence intensity in \u003cem\u003eNPM1\u003c/em\u003e mutant versus wild type cells, as well as the proportions of p53 expression in \u003cem\u003eNPM1\u003c/em\u003e-mutated cells between complete remission with or without complete count recovery (CR/CRi) and persistent disease (PD) groups. A p-value of less than 0.05 was considered statistically significant. The same p-value was used to assess differential gene expression; no adjustment was performed as only \u003cem\u003eTP53\u003c/em\u003e values were assessed.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eWild Type-TP53 is more highly expressed in NPM1-mutated AML compared to other AML subtypes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn our clinical diagnostic hematopathology practice we routinely perform screening for \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNPM1\u003c/em\u003e mutations by chromogenic immunohistochemistry (IHC) as a component of new AML evaluations.\u003csup\u003e8,10\u003c/sup\u003e In select cases of molecularly-\u003c/p\u003e\n\u003cp\u003econfirmed \u003cem\u003eNPM1\u003c/em\u003e-mutated/\u003cem\u003eTP53\u003c/em\u003e-WT AML, we have observed p53 expression at a level often observed in the cases harboring missense mutations in the DNA binding domain of \u003cem\u003eTP53\u003c/em\u003e (\u003cstrong\u003eFigure 1A, representative case\u003c/strong\u003e). We hypothesized that the elevated expression of WT-p53 could be a result of increased \u003cem\u003eTP53\u003c/em\u003e gene expression in at least a subset of \u003cem\u003eNPM1\u003c/em\u003e-AML cases. Based on an analysis of the BeatAML cohort dataset,\u003csup\u003e6\u003c/sup\u003e we found that \u003cem\u003eNPM1\u003c/em\u003e-AML cases (n=74) are associated with significantly higher \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003eexpression than \u003cem\u003eTP53\u003c/em\u003e-WT/\u003cem\u003eNPM1\u003c/em\u003e-WT cases (n=190) [p= 0.047, multivariate limma model] (\u003cstrong\u003eFigure 1B\u003c/strong\u003e). We considered the possibility that this difference may be driven by mutations with prognostic significance co-occurring with \u003cem\u003eNPM1\u003c/em\u003e (\u003cem\u003eFLT3\u003c/em\u003e-ITD, \u003cem\u003eFLT3\u003c/em\u003e-TKD, \u003cem\u003eDNMT3A\u003c/em\u003e, \u003cem\u003eSF3B1\u003c/em\u003e, \u003cem\u003eSRSF2\u003c/em\u003e, \u003cem\u003eU2AF1\u003c/em\u003e); however, exclusion of these genes by comparing cases of \u003cem\u003eNPM1\u003c/em\u003e-AML to \u003cem\u003eNPM1\u003c/em\u003e-WT/\u003cem\u003eTP53\u003c/em\u003e-WT/gene mutation-positive cases revealed a similar result\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;[p=0.018, multivariate limma model] (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). Furthermore, we observed no effect of specimen type (e.g., peripheral blood, bone marrow, or leukapheresis product) on \u003cem\u003eTP53\u003c/em\u003e expression level (\u003cstrong\u003eFigure 1D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep53-associated gene modules are upregulated in NPM1\u003c/em\u003e-\u003cem\u003emutated AML and correlate with wild-type TP53 expression level\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the activity of WT-p53 signaling we focused on known p53-associated gene sets. By comparing \u003cem\u003eNPM1\u003c/em\u003e-AML with \u003cem\u003eTP53\u003c/em\u003e-AML cases (n=17), we first established the pattern of up-/down-regulation for gene sets associated with DNA repair, apoptosis, and cell cycle pathways as a function of \u003cem\u003eTP53\u003c/em\u003e mutation (\u003cstrong\u003eFigure\u003c/strong\u003e \u003cstrong\u003e1E\u003c/strong\u003e). Despite the genetic heterogeneity among \u003cem\u003eNPM1\u003c/em\u003e-WT cases, relative to \u003cem\u003eNPM1\u003c/em\u003e-AML cases the -WT group exhibited a similar gene set enrichment pattern as\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eseen for \u003cem\u003eTP53\u003c/em\u003e-AML cases (\u003cstrong\u003eFigure 1F)\u003c/strong\u003e. We next performed a similar analysis restricted only to \u003cem\u003eNPM1\u003c/em\u003e-AML cases, comparing the uppermost (n=19) and lowermost (n=19) quartiles for \u003cem\u003eTP53\u003c/em\u003e gene expression; we noted a near-complete overlap in the pattern of up- and/or downregulation across DNA repair, apoptosis, and cell cycle gene sets as we observed when comparing \u003cem\u003eNPM1\u003c/em\u003e-AML and \u003cem\u003eTP53\u003c/em\u003e-AML cases (\u003cstrong\u003eFigure 1G\u003c/strong\u003e), suggesting that the activity of these pathways may be directly influenced by WT-p53 dosage within the context of \u003cem\u003eNPM1\u003c/em\u003e-AML.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWT-p53 protein is over-expressed in NPM1-mutated cells in AML and correlates with remission status post-induction therapy\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on our initial findings of increased WT-p53 protein expression in \u003cem\u003eNPM1\u003c/em\u003e-mutated cells in a case of AML, and evidence of increased \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003eexpression in \u003cem\u003eNPM1\u003c/em\u003e-AML cases within the BeatAML cohort, we further assessed p53 protein expression in a cohort of primary human bone marrow tissue samples from our institution (\u003cstrong\u003eTable 1\u003c/strong\u003e). Using a multiplex immunofluorescence-based phenotyping assay coupled with digital image analysis, we phenotyped cells in situ at single cell resolution, identifying them as \u003cem\u003eNPM1\u003c/em\u003e-mutant or -WT using a mutant protein-specific antibody, and then evaluated the frequency and intensity of p53 expression (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). The median number of nucleated cells analyzed per case was 8,957 (range: 1,869 - 31,151) [\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e]. Within each case, a greater proportion of \u003cem\u003eNPM1\u003c/em\u003e-mutated cells were p53-positive, compared to \u003cem\u003eNPM1\u003c/em\u003e-WT cells\u0026nbsp;[p\u0026lt;0.001, paired Wilcoxon test] (\u003cstrong\u003eFigure 2B-C\u003c/strong\u003e); overall, we found \u0026gt;10% of \u003cem\u003eNPM1\u003c/em\u003e\u0026shy;-mutant cells to be p53-positive in 24 of 33 cases (73%). Similarly, the mean fluorescence intensity (MFI) of p53 was higher in \u003cem\u003eNPM1\u003c/em\u003e-mutant compared to WT cells\u0026nbsp;[p\u0026lt;0.001, paired Wilcoxon test] (\u003cstrong\u003eFigure 2D-E\u003c/strong\u003e). We observed no significant difference in p53 proportion between patients above or below the age of 60, in those presenting with or without leukocytosis, or with respect to peripheral blood or bone marrow blast percentage (data not shown). We considered the possibility that p53 expression may simply be associated with a non-G0 state of the leukemic cells; however, we observed no correlation between p53 and Ki67 expression (\u003cstrong\u003eSupplemental Figure 2\u003c/strong\u003e). We also wondered if p53 overexpression could be a result of diminished MDM2-mediated degradation due to cytoplasmic sequestration of MDM2 by mutant NPM1; however, an analysis of the few highest p53 co-expressors revealed no significant cytoplasmic MDM2 signal by multiplex immunofluorescence (\u003cstrong\u003eSupplemental Figure 3\u003c/strong\u003e). Furthermore, we found no significant difference in p53 co-expression frequency based on presence or absence of common co-mutations (e.g. \u003cem\u003eFLT3\u003c/em\u003e-ITD, \u003cem\u003eDNMT3A\u003c/em\u003e, \u003cem\u003eIDH1/2\u003c/em\u003e) [\u003cstrong\u003eSupplemental Figure 4\u003c/strong\u003e]. As a proportion of total nucleated cells, p53 was most frequently detected in \u003cem\u003eTP53\u003c/em\u003e-AML cases, as anticipated; \u003cem\u003eNPM1\u003c/em\u003e-AML cases included a range of p53 expression frequency, with a subset exhibiting p53 expression near the level found in \u003cem\u003eTP53\u003c/em\u003e-AML cases. We observed no significant difference in p53 co-expression among total nucleated cells between \u003cem\u003eNPM1\u003c/em\u003e-AML and a small comparison group of normal karyotype \u003cem\u003eNPM1\u003c/em\u003e-WT cases (p\u0026gt;0.05) [\u003cstrong\u003eSupplemental Figure 5\u003c/strong\u003e]. Similarly, p53 MFI was significantly higher in \u003cem\u003eTP53\u003c/em\u003e-mutated versus all \u003cem\u003eTP53\u003c/em\u003e-WT cases (\u003cstrong\u003eSupplemental Figure 6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePost-induction remission status was available for 27 of the 33 cases analyzed. Interestingly, we found a significantly higher p53-positive proportion among \u003cem\u003eNPM1\u003c/em\u003e-mutated cells at diagnosis in patients who achieved complete remission with or without complete count recovery (CR/CRi, n=21) compared to patients with grossly persistent disease (PD, n=6) [median 0.219 vs. 0.086, p=0.018] [\u003cstrong\u003eFigure 2F\u003c/strong\u003e]. Of note, we found no difference in the frequency of \u003cem\u003eFLT3\u003c/em\u003e-ITD co-mutations in CR/CRi versus PD patients (p\u0026gt;0.05).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe basis for many of the biological (e.g. normal karyotype/genomic stability) and clinical (e.g. favorable response to induction therapy) features of \u003cem\u003eNPM1\u003c/em\u003e-AML remains largely unknown. While many patients have a favorable clinical course, a subset experience recurrent disease after a period of remission, or more rarely, refractory disease after initial therapy. Many, but not all, patients with this trajectory can be anticipated using well-recognized adverse clinical and/or genetic features, although other biomarkers with predictive potential remain to be identified.\u003c/p\u003e \u003cp\u003eIn stark contrast, \u003cem\u003eTP53\u003c/em\u003e-AML is associated with uniformly dismal outcomes; and interestingly, \u003cem\u003eNPM1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e mutations are considered essentially mutually exclusive.\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Through routine use of protein immunohistochemistry to assess p53 and mutant NPM1 protein expression status in the diagnostic evaluation of AML cases, we observed cases of \u003cem\u003eNPM1\u003c/em\u003e-AML with a high frequency and intensity of WT-p53 expression, leading us to hypothesize that the common clinical and biological features of \u003cem\u003eNPM1\u003c/em\u003e-AML could be explained, at least in part, by retained WT-p53 activity.\u003c/p\u003e \u003cp\u003eAn exploration of publicly available RNA sequencing data from the Beat AML cohort revealed higher \u003cem\u003eTP53\u003c/em\u003e gene expression in \u003cem\u003eNPM1\u003c/em\u003e-AML cases compared to \u003cem\u003eNPM1\u003c/em\u003e-WT AML cases. Of note, we observed no difference in \u003cem\u003eTP53\u003c/em\u003e expression level among \u003cem\u003eNPM1\u003c/em\u003e-AML cases upon separating them based on mutational status of genes with known prognostic significance (e.g. \u003cem\u003eFLT3\u003c/em\u003e-ITD, \u003cem\u003eDNMT3A\u003c/em\u003e). Given recently published data demonstrating that mutant NPM1 protein directly binds to chromatin to modify gene expression,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e we explored the possibility that mutant NPM1 could be directly driving \u003cem\u003eTP53\u003c/em\u003e expression, but did not find compelling evidence to support this mechanism. We then used gene set enrichment analysis to evaluate the functional status of p53, focusing primarily on pathways related to DNA repair, apoptosis, and cell cycle regulation. We observed an overlapping pattern of positively or negatively enriched gene sets for \u003cem\u003eNPM1\u003c/em\u003e-AML cases when compared to either \u003cem\u003eTP53\u003c/em\u003e-AML or \u003cem\u003eNPM1\u003c/em\u003e-WT cases. Interestingly, separating \u003cem\u003eNPM1\u003c/em\u003e-AML cases into those with high and low \u003cem\u003eTP53\u003c/em\u003e co-expression recapitulated the gene set enrichment differences observed between \u003cem\u003eNPM1\u003c/em\u003e-AML and \u003cem\u003eTP53\u003c/em\u003e-AML cases; this result suggests that low \u003cem\u003eTP53\u003c/em\u003e co-expression in \u003cem\u003eNPM1\u003c/em\u003e-AML may approximate the absence of a normally functioning p53 protein.\u003c/p\u003e \u003cp\u003eWe finally assessed p53 protein expression in a cohort of patient samples from our institution using a multiplex immunofluorescence assay with digital image analysis, capitalizing on the concurrent application of a mutant NPM1 protein-specific antibody to define the mutational status of individual cells. These studies revealed elevated p53 expression in \u0026gt;\u0026thinsp;10% of \u003cem\u003eNPM1\u003c/em\u003e-mutant cells in 73% of the analyzed cases, with a higher frequency and intensity of p53 expression in \u003cem\u003eNPM1\u003c/em\u003e mutant relative to WT cells observed in all cases. Interestingly, patients with grossly persistent disease following induction therapy were characterized by a significantly lower frequency of p53 expression at diagnosis than those who achieved complete remission.\u003c/p\u003e \u003cp\u003eOur study is limited by its retrospective design, the small internal cohort of patient samples studied, and largely correlative findings. However, to the best of our knowledge, our data provide the first evidence that many \u003cem\u003eNPM1\u003c/em\u003e-AML cases are characterized by elevated \u003cem\u003eTP53\u003c/em\u003e gene and p53 protein expression at baseline. It remains possible that this profile could be driven by either 1) MDM2 sequestration by residual WT-NPM1 protein with consequently reduced MDM2-mediated p53 degradation, or 2) p53 stabilization via phosphorylation at select serine residues.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Although mechanistic studies will be required to explore these and other potential mechanisms underlying increased p53 expression, we present evidence suggesting a role for increased \u003cem\u003eTP53\u003c/em\u003e gene expression; this finding is associated with activity of p53-mediated gene expression pathways linked to DNA repair, apoptosis, and cell cycle regulation opposite that observed in \u003cem\u003eTP53\u003c/em\u003e-AML. We therefore hypothesize that, while increased p53 activity might contribute to the intrinsic biological and clinical features of most \u003cem\u003eNPM1\u003c/em\u003e-AML cases, low p53 expression in a subset of cases at diagnosis could represent a potential biomarker of unfavorable disease worthy of further exploration in larger cohorts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eExperiments were designed, executed and/or analyzed by P.B., I.V., A.S.K., C.R.C., C.M., M.G., J.K., J.A.F., C.E.M., and S.S.P. I.V. performed multiplex tissue staining and imaging. J.K., J.A.F., G.J.R. and P.D. reviewed clinical data. The manuscript was written by P.B. and S.S.P. with input from all authors. P.B. and S.S.P. conceived of and designed the study. S.S.P. supervised the work.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis work was supported by the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College (start-up funding to S.S.P.). C.R.C. is supported by an NIH post-doctoral training grant (T32AR071302). Tissue staining and imaging was performed in the Multiparametric In Situ Imaging (MISI) Laboratory of the Department of Pathology and Laboratory Medicine, Weill Cornell Medical College.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eAnalysis code and data are available upon reasonable written request to the corresponding author (e-mail: [email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia. 2022;36(7):1703\u0026ndash;1719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArber DA, Orazi A, Hasserjian RP, et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022;140(11):1200\u0026ndash;1228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalini B, Brunetti L, Sportoletti P, Martelli MP. NPM1-mutated acute myeloid leukemia: from bench to bedside. Blood. 2020;136(15):1707\u0026ndash;1721.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOthman J, Potter N, Ivey A, et al. Molecular, clinical, and therapeutic determinants of outcome in NPM1-mutated AML. Blood. 2024;144(7):714\u0026ndash;728.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapaemmanuil E, Gerstung M, Bullinger L, et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N. Engl. J. Med. 2016;374(23):2209\u0026ndash;2221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyner JW, Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526\u0026ndash;531.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasqualucci L, Liso A, Martelli MP, et al. Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: Impact on WHO classification. Blood. 2006;108(13):4146\u0026ndash;4155.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel SS, Pinkus GS, Ritterhouse LL, et al. High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia. Am. J. Hematol. 2019;94(8):921\u0026ndash;928.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez A, Patel S, Geyer JT, et al. Comparison of Multiple Clinical Testing Modalities for Assessment of NPM1-Mutant AML. Front. Oncol. 2021;11:701318.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTashakori M, Kadia T, Loghavi S, et al. TP53 copy number and protein expression inform mutation status across risk categories in acute myeloid leukemia. Blood. 2022;140(1):58\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 2005;102(43):15545\u0026ndash;15550.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarachakov A, Varlamova A, Svelolkin V, et al. Spatial Mapping of Human Hematopoiesis at Single Cell Resolution Reveals Aging-Associated Topographic Remodeling. \u003cem\u003eBlood\u003c/em\u003e. 2023;blood.2023021280.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUckelmann HJ, Haarer EL, Takeda R, et al. Mutant NPM1 directly regulates oncogenic transcription in acute myeloid leukemia. Cancer Discov. 2022;CD-22-0366.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang XQD, Fan D, Han Q, et al. Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia. Cancer Discov. 2023;13(3):724\u0026ndash;745.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshcroft M, Kubbutat MH, Vousden KH. Regulation of p53 function and stability by phosphorylation. Mol. Cell. Biol. 1999;19(3):1751\u0026ndash;1758.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"blood-cancer-journal","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"bcj","sideBox":"Learn more about [Blood Cancer Journal](http://www.nature.com/bcj/)","snPcode":"41408","submissionUrl":"https://mts-bcj.nature.com/cgi-bin/main.plex","title":"Blood Cancer Journal","twitterHandle":"@bloodcancerjnl","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NPM1, AML, p53, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-5214655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5214655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSomatic mutations in nucleophosmin (\u003cem\u003eNPM1\u003c/em\u003e) are key in defining a common subtype of acute myeloid leukemia (\u003cem\u003eNPM1\u003c/em\u003e-AML), characterized by chromosomal stability and favorable therapeutic responses. However, some patients exhibit a suboptimal response to initial treatment, and relapses are common, highlighting the need for novel biomarkers. Notably, \u003cem\u003eNPM1\u003c/em\u003eand \u003cem\u003eTP53\u003c/em\u003e mutations rarely co-occur in AML, where \u003cem\u003eTP53\u003c/em\u003e mutations correlate with aneuploidy and poor survival outcomes. Here, we present transcriptomic and proteomic evidence of unexpectedly high wild-type (WT) \u003cem\u003eTP53\u003c/em\u003e/p53 expression in a subset of \u003cem\u003eNPM1\u003c/em\u003e-AMLs at baseline. Analyses of the Beat AML cohort indicate that \u003cem\u003eNPM1\u003c/em\u003e-AMLs generally express more \u003cem\u003eTP53\u003c/em\u003ethan \u003cem\u003eNPM1\u003c/em\u003e/\u003cem\u003eTP53\u003c/em\u003e-WT AMLs. Among seventy-four \u003cem\u003eNPM1\u003c/em\u003e-AML samples, those with the lowest \u003cem\u003eTP53\u003c/em\u003e co-expression are enriched for downregulated signatures linked to DNA repair, apoptosis, and the cell cycle. By profiling thirty-three primary patient samples using multiplex immunofluorescence staining with single cell-based quantitative digital image analysis, we observed increased p53 expression in \u003cem\u003eNPM1\u003c/em\u003e-mutant cells compared to WT cells. Importantly, patients with primary refractory disease showed low p53 co-expression at diagnosis. These findings suggest that WT-p53 might play a crucial role in the biological and clinical characteristics seen in \u003cem\u003eNPM1\u003c/em\u003e-AML.\u003c/p\u003e","manuscriptTitle":"Wild-Type p53 Overexpression in NPM1-Mutated AML: Potential Implications for Disease Biology and Therapy Response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-09 16:48:33","doi":"10.21203/rs.3.rs-5214655/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Blood Cancer Journal","date":"2024-12-04T15:52:04+00:00","index":"","fulltext":""},{"type":"decision","content":"Reject after peer review","date":"2024-12-03T09:55:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-05T10:13:45+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-10-23T13:37:44+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-10-20T07:24:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-10-08T12:49:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-08T10:12:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-08T10:12:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2024-10-08T10:02:33+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-10-07T10:29:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"blood-cancer-journal","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"bcj","sideBox":"Learn more about [Blood Cancer Journal](http://www.nature.com/bcj/)","snPcode":"41408","submissionUrl":"https://mts-bcj.nature.com/cgi-bin/main.plex","title":"Blood Cancer Journal","twitterHandle":"@bloodcancerjnl","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"157cb5e8-981d-437b-a7d8-f048d7d8387a","owner":[],"postedDate":"October 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":38750802,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"},{"id":38750803,"name":"Health sciences/Pathogenesis/Oncogenesis"},{"id":38750804,"name":"Health sciences/Medical research/Translational research"},{"id":38750805,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"}],"tags":[],"updatedAt":"2024-12-11T21:40:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-09 16:48:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5214655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5214655","identity":"rs-5214655","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}