BMT for Myelodysplastic Syndrome: When and Where and How

Myelodysplastic syndromes (MDS) are a diverse group of hematological malignancies distinguished by a combination of dysplasia in the bone marrow, cytopenias and the risk of leukemic transformation. The hallmark of MDS is bone marrow failure which occurs due to selective growth of somatically mutated clonal hematopoietic stem cells. Multiple prognostic models have been developed to help predict survival and leukemic transformation, including the international prognostic scoring system (IPSS), revised international prognostic scoring system (IPSS-R), WHO prognostic scoring system (WPSS) and MD Anderson prognostic scoring system (MDAPSS). This risk stratification informs management as low risk (LR)-MDS treatment focuses on improving quality of life and cytopenias, while the treatment of high risk (HR)-MDS focuses on delaying disease progression and improving survival. While therapies such as erythropoiesis stimulating agents (ESAs), erythroid maturation agents (EMAs), immunomodulatory imide drugs (IMIDs), and hypomethylating agents (HMAs) may provide benefit, allogeneic blood or marrow transplant (alloBMT) is the only treatment that can offer cure for MDS. However, this therapy is marred, historically, by high rates of toxicity and transplant related mortality (TRM). Because of this, alloBMT is considered in a minority of MDS patients. With modern techniques, alloBMT has become a suitable option even for patients of advanced age or with significant comorbidities, many of whom who would not have been considered for transplant in prior years. Hence, a formal transplant evaluation to weigh the complex balance of patient and disease related factors and determine the potential benefit of transplant should be considered early in the disease course for most MDS patients. Once alloBMT is recommended, timing is a crucial consideration since delaying transplant can lead to disease progression and development of other comorbidities that may preclude transplant. Despite the success of alloBMT, relapse remains a major barrier to success and novel approaches are necessary to mitigate this risk and improve long term cure rates. This review describes various factors that should be considered when choosing patients with MDS who should pursue transplant, approaches and timing of transplant, and future directions of the field.

IRF4 expression is low in Philadelphia negative myeloproliferative neoplasms and is associated with a worse prognosis

Interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various hematologic malignancies. Its expression has been related to the negative regulation of myeloid-derived suppressor cells (MDSCs) and the polarization of anti-inflammatory M2 macrophages, thereby altering immunosurveillance and inflammatory mechanisms. An abnormal inflammatory status in the bone marrow microenvironment of myeloproliferative neoplasms (MPNs) has recently been demonstrated; moreover, in chronic myeloid leukemia a downregulated expression of IRF4 has been found. In this context, we evaluated the IRF4 expression in 119 newly diagnosed consecutive Philadelphia negative MPNs (Ph- MPNs), showing a low expression among the MPNs phenotypes with a more significant decrease in primary myelofibrosis patients. Lower IRF4 levels were associated with JAK2 + and triple negatives cases carrying the worst prognosis. Furthermore, the IRF4 levels were related to leukemic transformation and a shorter leukemia-free survival; moreover, the risk of myelofibrosis transformation in polycythemia vera and essential thrombocythemia patients was more frequent in cases with lower IRF4 levels. Overall, our study demonstrates an IRF4 dysregulated expression in MPNs patients and its association with a worse prognosis. Further studies could validate these data, to improve our knowledge of the MPNs pathogenesis and confirm the IRF4 role as a new prognostic factor.

Atypical Chronic Myelogenous Leukemia, BCR-ABL1 Negative: Diagnostic Criteria and Treatment Approaches

Atypical chronic myelogenous leukemia (aCML), BCR/ABL1 negative is a rare myelodysplastic/myeloproliferative neoplasm, usually manifested with hyperleukocytosis without monocytosis or basophilia, organomegaly, and marked dysgranulopoiesis. In this review, we will discuss the classification and diagnostic criteria of aCML, as these have been formulated during the past 30 years, with a focus on the recent advances in the molecular characterization of the disease. Although this entity does not have a definitive molecular profile, its molecular characterization has contributed to a better understanding and more accurate classification and diagnosis of aCML. At the same time, it has facilitated the identification of adverse prognostic factors and the stratification of patients according to their risk for leukemic transformation. What is more, the molecular characterization of the disease has expanded our therapeutic choices, thoroughly presented and analyzed in this review article.

Cytogenetics in Essential Thrombocythemia: Phenotype and Molecular Correlates and Prognostic Relevance in 818 Informative Cases

Background Cytogenetic abnormalities at diagnosis are relatively uncommon in essential thrombocythemia (ET). In the current study of 818 consecutive patients with ET who were fully annotated for karyotype, we describe the spectrum and prevalence of cytogenetic abnormalities at diagnosis, followed by a comprehensive assessment of phenotypic and molecular correlates and prognostic relevance. Methods The study cohort consisted of 818 consecutive patients with ET that were diagnosed according to the World health Organization 2016 criteria and underwent evaluation between 1967-2021. In order to minimize the inadvertent inclusion of patients with masked polycythemia vera, JAK2 mutated cases with hemoglobin (Hb) level >16 g/dL in women and 16.5 g/dL in men were excluded; similarly, cases with anemia defined by sex adjusted Hb level of <11 g/dL in women and <12.5 g/dL in men were also excluded, in order to avoid inadvertent inclusion of patients with prefibrotic myelofibrosis. Cytogenetic studies were performed either at or within one year of diagnosis and reported according to the International System for Human Cytogenetic Nomenclature. Disease status and survival information was updated in May 2021. JMP Pro 16.0.0 software package, SAS Institute, Cary, NC was utilized for all analyses. Results Prevalence and spectrum of cytogenetic abnormalities Karyotype was normal in 755 patients (92%), showed loss of Y chromosome (-Y) in 16 (2%), and showed abnormalities other than -Y in 47 (5.7%); most common abnormalities included del(20q) (n=10, 21%), trisomy 9 (n=8, 17%), trisomy 8 (n=2, 4%), del(5q) (n=2, 4%), and del(3p) (n=2, 4%). Other sole cytogenetic abnormalities were identified in 18 (38%) patients. Phenotypic and molecular correlates Abnormal karyotype, other than -Y, in comparison with normal karyotype was associated with older age (median age; 63 vs 58 years, p=0.02), lower hemoglobin level (p=0.003), and a higher incidence of arterial thrombosis prior to/at diagnosis (25% vs 13%; p=0.03). 603 patients were annotated for driver mutations; abnormal/normal/-Y frequencies were 78%/60%/71% for JAK2, 22%/26%/14% CALR, 0%/3%/0% MPL and 0%/10% /14% triple negative (p=0.31). NGS information was available in 226 patients and showed absence of ASXL1 mutation in all patients with abnormal karyotype vs 8/211 (4%) with normal karyotype vs 2/4 (50%) with -Y (p<0.0001). Disease transformation and overall-survival. At a median follow-up of 9.6 years (range; 0.01-49.4 years), a total of 96 patients (12%) underwent fibrotic transformation: 6 (13%) with abnormal karyotype, 89 (12%) with normal karyotype and 1 (6%) with -Y (p=0.77). Leukemic transformation rates were also similar with respective frequencies of 4%, 3% and 0% (p=0.71). Abnormal karyotype and -Y were associated with inferior survival with median of 12 years (range; 0.1-34) and 9 years (range; 0.01- 19.9), respectively, compared to 21 years (range; 0.01-49.4) for normal karyotype (p<0.0001) (Figure). In univariate analysis, risk factors for overall survival included abnormal karyotype (p=0.001), - Y (p=0.004), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p<0.0001), male gender (p=0.0003), and history of thrombosis (p=0.001). During multivariable analysis, abnormal karyotype other than -Y (p=0.003), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p=0.001), and male gender (p=0.01) remained significant. Additional analysis suggested individual prognostic impact for del(20q) (p=0.04) and also for trisomy 9 (p=0.09) and other abnormalities (p=0.07), with borderline significance. Conclusion The current study confirms the association of abnormal karyotype in ET with older age, lower hemoglobin level, and history of arterial thrombosis, and its mutual exclusivity with ASXL1 mutations. Our observation regarding the independent adverse impact of abnormal karyotype other than -Y, on overall survival, in the absence of association with fibrotic or leukemic transformation, requires clarification from additional studies, which should also investigate the effect of specific abnormalities. Figure 1 Figure 1. Disclosures Szuber: Novartis: Honoraria.

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation and Immune Attack

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. Individuals with MPN are at increased risk for transformation to bone marrow fibrosis (myelofibrosis, MF) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. However, targetable mechanisms underlying progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in stem cells and aberrantly overexpressed in aggressive tumors (Xian et al Nature Commun 2017;8:15008, Resar et al Cancer Res 2018;78:1890). Transgenic mice misexpressing Hmga1 in lymphoid cells develop lethal leukemia by dysregulating gene networks associated with aberrant proliferation and inflammation (Hillion et al Cancer Res 2008;68:10121, Schuldenfrei et al BMC Genomics, 2011;12:549). We discovered that HMGA1 is overexpressed in MPN with progression and required for leukemic transformation in preclinical models (Resar et al Blood 2018;132 Suppl 1:102). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy. Methods: To elucidate transcriptional networks regulated by HMGA1 during MPN progression to AML, we integrated multi-omics sequencing (seq) analyses, including RNAseq, chromatin immunoprecipitation seq (ChIPseq), and ATACseq in AML cell lines from JAK2 V617Fmutant MPN after leukemic transformation (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNA (shRNA)-mediated gene silencing. Gene set enrichment analysis was used to dissect molecular mechanisms underlying immune invasion by HMGA1. To validate results in human MPN, RNAseq was performed in peripheral blood mononuclear cells (PBMCs) from matched MF patients who transformed to AML. To reconstruct the immune cell composition of primary MPN samples, we applied xCell, a robust computational method that converts gene expression profiles to immune cell types. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays. Results: Integration of RNAseq, ChIPseq, and ATACseq in MPN AML cells (DAMI, SET-2) revealed that HMGA1 represses genes involved in immune activation (inflammatory response, TNFa signaling, NF-κB networks) and antigen presentation [Interferon gamma (IFNγ) response networks], including genes encoding the major histocompatibility complex (MHC) class I and II antigens. Inhibiting HMGA1 results in up-regulation of MHC class I and II antigen genes, with greatest induction of HLA-DRA (the alpha paralog for HLA Class II antigens). Similarly, HMGA1 depletion increases cell surface expression of HLA-DRA antigens. Strikingly, RNAseq from MPN patients with MF after transformation to AML reveal that HMGA1 is up-regulated in MPN AML concurrent with repression in gene networks of immune activation and antigen presentation. Immune cell transcriptomes are also depleted in MPN cells after leukemic transformation. To determine if immune evasion by HMGA1 could be modulated in therapy, we applied CMAP which identified histone deacetylase inhibitors (HDACi) as drugs targeting the HMGA1 transcriptome. The HDACi, entinostat, is cytotoxic and synergizes with the JAK inhibitor, ruxolitinib, in MPN AML cell lines. Further, entinostat induces expression of MHC class II genes and antigens. Moreover, HMGA1 depletion enhances sensitivity of MPN AML cells to entinostat. Conclusions: We discovered an epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and enhancing chromatin accessibility to activate transcriptional networks that repress antigen presentation and immune attack. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN and can be targeted by HDACi therapy. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks could activate an immune attack and prevent MPN progression. Figure 1 Figure 1. Disclosures Rampal: Novartis: Consultancy; Pharmaessentia: Consultancy; Kartos: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Sierra Oncology: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Incyte: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment.

Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)

Background: Clonal evolution in AML originates long before diagnosis and is a highly dynamic process. Having a greater understanding of leukemogenesis may contribute to develop treatment strategies that target the tumor evolutionary process. However, dissecting leukemic transformation at the onset of AML is challenging without single-cell sequencing, and most clinical laboratories do not have infrastructure to perform these studies routinely. Patients with newly diagnosed AML may present dysplasia. If these residual, mature, dysplastic cells were generated before the differentiation blockage of blasts preceding leukemic transformation, it could be hypothesized that studying the genetic landscape of dysplastic cells and blasts could uncover the evolutionary process from dysplasia to AML. This hypothesis has never been investigated. Aim: Reconstruct clonal evolution from dysplasia to AML based on the genetic signature of dysplastic cells and leukemic blasts, analyzed using integrated MFC immunophenotyping and sorting with NGS. Methods: Presence of dysplasia according to aberrant phenotypic differentiation of the neutrophil, monocytic and erythroid lineages was investigated using MFC and EuroFlow MDS/AML panels in 283 newly diagnosed AML patients (median age 74; range 29-90). Patient-specific phenotypes were leveraged to isolate a total of 99 cell types from 22 AML cases for targeted (48 MDS/AML related genes) and whole-exome sequencing (WES), with a mean depth of 3246x and 141x, respectively. In patients with measurable residual disease (MRD) by MFC at the time of complete remission, tumor resistant cells were FACSorted for WES using patient-specific aberrant phenotypes. T cells were used as germline control in both approaches. Mutations were considered if ≥0.05 allele frequency in leukemic blasts or dysplastic cells and ≤0.2 in T cells. Results: We first assessed the applicability of our hypothesis by investigating how many patients show dysplasia at the onset of AML. Dysplastic cells were observed in 252 of 283 (89%) cases. Phenotypic abnormalities were more frequently noted in the neutrophil lineage (47%), followed by the monocytic (40%) and erythroid cells (13%). Up to 169/283 (60%) patients showed multi-lineage dysplasia. Only nine cases showed no signs of dysplasia, whereas the remaining 22 had undetectable hematopoiesis. Targeted sequencing of dysplastic cells and blasts in 16 patients uncovered three evolutionary patterns of leukemogenesis. Stable transition in those displaying identical mutational landscapes in blasts and residual mature dysplastic cells (9/16); clonal selection in cases where blasts originated from leukemic stem cells other than the ones driving dysplasia, due to mutations absent in blasts and present in dysplastic cells (4/16); and clonal evolution in cases showing new mutations in blasts onto mutations shared between these and dysplastic cells (3/16). Interestingly, most patients displaying stable transition from dysplasia to AML had mutated ASXL1, RUNX1 and/or TP53 (8/9). Mutations present in dysplastic cells while absent in blasts from patients showing a clonal selection evolutionary pattern, were more frequently detected in genes related to signaling pathways (eg JAK2, KRAS and NRAS). By contrast, clonal evolution was characterized by new mutations affecting FLT3ITD and STAG2. The higher throughput of WES of dysplastic cells and blasts from six patients unveiled a more complex dynamic process of leukemogenesis, with all three evolutionary patterns being detectable in nearly all cases. Most interestingly, we found patients with mutations in dysplastic cells and blasts at diagnosis, but not in MRD cells (eg NBPF1 and ZNF717); and patients showing mutations in dysplastic and MRD cells, but not in blasts at diagnosis (eg MUC2 and KIR2DL3). These findings uncover that genetic alterations that are critical in leukemic transformation and chemoresistance, may not overlap (Figure). Conclusions: We showed for the first time that it is possible to reconstruct leukemogenesis in nearly 90% of newly-diagnosed AML patients, using techniques that are commonly available in clinical laboratories. The possibility to identify the genetic drivers of leukemic transformation and chemoresistance, could be clinically meaningful to develop tailored treatment strategies aiming at the eradication of genetically diverse leukemic clones. Figure 1 Figure 1. Disclosures Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding. Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria. Perez-Simon: JANSSEN, TAKEDA, PFIZER, JAZZ, BMS, AMGEN, GILEAD: Other: honorarium or budget for research projects and/or participation in advisory boards and / or learning activities and / or conferences. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Montesinos: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline/Menarini: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Consultancy; Tolero Pharmaceutical: Consultancy; Forma Therapeutics: Consultancy; Glycomimetics: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Paiva: Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy.

The BMP/SMAD Pathway Is a Key Mediator of Leukemic Transformation of TP53-Mutant Post-MPN AML

Leukemic transformation (LT) after an antecedent myeloproliferative neoplasm (MPN) carries a dismal prognosis. As such, there is a pressing need for new mechanistic insights into LT as well as novel therapeutic approaches. Mutational inactivation of TP53 is the most common somatic mutation in LT. However, the impact of TP53 allelic state on the ability to potentiate LT, as well as the pathways involved in this process, have largely remained unresolved. To investigate the role of Tp53 alterations in LT, we generated an allelic series of mouse models with Jak2V617F/+ combined with conditional Tp53 knockout and point mutant alleles (all crossed to Rosa-CreERT2); Jak2V617F/+(J VF) , Jak2V617F/+-Tp53fl/+(J VFP +/-), Jak2V617F/+-Tp53fl/fl (J VFP -/-), Jak2V617F/+-Tp53R172H/+(J VFP R172H/+), Jak2V617F/+-Tp53R172H/fl (J VFP R172H/-). After tamoxifen-induced recombination, mice transplanted with J VF, J VFP +/- and J VFP R172H/+ cells developed an MPN phenotype, whereas all the recipients of J VFP -/- and J VFP R172H/- bone marrow initially developed an MPN phenotype followed by transformation to acute leukemia with significantly impaired survival, and changes in blood counts and organ weights, compared to other genotypes (Fig 1A/B). Histopathology of J VFP -/- and J VFP R172H/- mice was consistent with pure erythroleukemia (PEL; Fig 1C). Analysis of stem and progenitor compartments demonstrated that the MEP (Megakaryocyte Erythroid Progenitors) compartment was significantly expanded in the bone marrow and spleen of both J VFP -/- and J VFP R172H/- mice, compared to other genotypes, at both the MPN and PEL stages of disease, consistent with erythroid-biased hematopoiesis (Fig 1D). Given we observed sequential MPN->AML progression, we hypothesized that additional genetic/biological events were required to promote LT. Sparse whole genome sequencing analysis revealed that transformation to PEL was associated with the development of recurrent copy number alterations (CNA) . Importantly, CNAs were restricted to the MEP compartment and not identified in the GMP compartment (Fig 1E), suggesting that MEPs might represent the leukemia initiating population with capability of acquiring additional genomic instability. Consistent with this hypothesis, mice transplanted with MEPs, but not GMPs from J VFP -/- and J VFP R172H/- mice at the MPN stage developed PEL. Further, single-cell RNA sequencing of J VF and J VFP -/- (at both MPN and PEL stage) demonstrated that the gene-expression signature of the leukemic population was most similar to that of erythroid progenitors and erythroblasts, and that by copy number inference analysis, CNAs were restricted to the leukemic population. We identified 617 genes up-regulated in both J VFP -/- and J VFP R172H/- leukemic MEPs when compared to J VF MEPs using RNA-seq. Pathway analysis demonstrated increased expression of Bone morphogenetic protein (BMP) pathway genes in both J VFP -/- and J VFP R172H/- leukemic mice (Fig 1F). Importantly, similar observations were made in human PEL samples as well. To investigate the function of this pathway, leukemic MEPs from J VFP -/- and J VFP R172H/- mice were transduced with an shRNA-targeting Bmp2 or a control and injected into lethally irradiated recipient mice. Mice injected with Bmp2-shRNA MEPs demonstrated leukemic regression and restoration of normal hematopoiesis as evidenced by significant reductions in leukocytosis (p<0.05) and increased HGB (p<0.05) and an increase in PLT count (p<0.05/p<0.01) (Fig 1G). Finally, as compared to mice injected with leukemic MEPs with control shRNA, mice injected with Bmp2-shRNA had significantly longer survival (p<0.05) (Fig 1H). Thus, downregulation of Bmp2 results in attenuation of the leukemic phenotype. Using novel models, we have identified that bi-allelic, but not mono-allelic Tp53 alteration is required for LT of MPN. The leukemia initiating population arises within the MEP compartment and is characterized by recurrent CNAs acquired in a specific hematopoietic compartment. Moreover, the BMP/SMAD pathway is upregulated in leukemic MEPs and plays a functional role in LT. Collectively, our data yields novel biological insights into the process of leukemic transformation mediated by Tp53 alterations. Data on selective therapeutic targeting of p53-mutant PEL will be presented at the meeting. Figure 1 Figure 1. Disclosures Xiao: Stemline Therapeutics: Research Funding. Lowe: Oric Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Mirimus, Inc: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Faeth Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; PMV Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Janssen: Consultancy; Astellas: Consultancy. Rampal: Pharmaessentia: Consultancy; Abbvie: Consultancy; Kartos: Consultancy; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Novartis: Consultancy; CTI: Consultancy; Sierra Oncology: Consultancy; Stemline: Consultancy, Research Funding; Blueprint: Consultancy; Memorial Sloan Kettering: Current Employment.

Decision Analysis of Allogeneic Stem Cell Transplantation for Primary Myelofibrosis

Introduction: Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by cytopenias, splenomegaly and a risk of leukemic transformation. In light of newer therapies such as ruxolitinib that are not curative but can improve quality of life, the timing of transplant needs more in-depth analysis to determine which patients would benefit from an early versus delayed transplant strategy. Methods: We developed a Markov cohort model to simulate the long-term disease trajectory in patients with PMF and predict the optimal timing of transplant stratified by a Dynamic International Prognostic Scoring System (DIPSS) risk. Our model consisted of five health states including alive with PMF, alive after leukemic transformation, alive after transplant, alive after relapse and death. Transition probabilities between health states were acquired from published literature on the natural history of the disease and outcomes following transplantation. The model was run over a patient's lifetime until all patients transitioned to the death state. We used a cycle length of one-month to represent the natural progression of PMF. The structure of the Markov model is delineated in Figure 1. In this decision model, a hypothetical cohort of patients begins in the Alive-PMF state and can transition after each monthly cycle to other health states. Patients could remain in an alive state for any number of cycles without transitioning to another health state, indicated by the arrow wheels. We performed probabilistic analyses by jointly varying all model parameters over 1000 simulations and calculated 95% confidence intervals (CI) for the model outcome. Results: Regardless of DIPSS risk, all patients with PMF benefited from a transplant with respect to life expectancy gained (Figure 2). Life expectancy gains from a transplant among patients with high-risk disease peak at 9.7 months (95% CI: 9.5-9.9) from diagnosis, while patients with intermediate-2 disease have a peak gain in life expectancy at 16.6 months (95% CI: 16.4-16.8). Intermediate-1 DIPSS risk patients have a more delayed time frame where the net gain in life expectancy from transplant begins to slow at 20.5 months (95% CI: 20.2-20.7). Patients with low risk DIPSS had greater net gain in life expectancy the longer transplant was delayed; this trend plateaued at 29 to 45 months, when thereafter net gain in life expectancy begins to be lost (Figure 1). Conclusion: Our modeling suggests that transplant processes including donor selection and pre-transplant work-up are indicated upfront for patients diagnosed with intermediate-2 and high risk PMF, while this can be delayed for patients with low or intermediate-1 risk disease. This model should provide clinicians with guidance on when to refer eligible patients with PMF for transplantation. Figure 1 Figure 1. Disclosures Kekre: Novartis: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy. Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP). Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617Fmurine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression. Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. Figure 1 Figure 1. Disclosures Rampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.