MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia by Enhancing Common β Chain Cytokine Receptor Endocytosis

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells, but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase. Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor, but promotes endocytosis of common β chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhance leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and blast phase CML. Promoting MS4A3 re-expression or delivery of ectopic MS4A3 may help eliminating LSPCs in vivo.

Clinico-Molecular Features and Treatment Outcomes in Primary Myelofibrosis Phenotypes with Protracted Disease Dynamics

Background Primary myelofibrosis (PMF) is the most aggressive subtype among classical BCR-ABL1 negative myeloproliferative neoplasms (MPN). Driven by constitutive activation of the JAK/STAT pathway, its prognosis is defined by cardinal clinical, cytogenetic and molecular features. While most patients require therapy for symptomatic splenomegaly, disease-related symptoms, or cytopenias, asymptomatic lower-risk patients may be appropriately monitored with active surveillance. The aim of this study was to explore disease characteristics and outcomes among pts who remained on prolonged active surveillance compared to those who received early treatment. Methods We identified patients with confirmed MF (inclusive of primary MF and MF occurring after essential thrombocythemia or polycythemia vera) treated at Moffitt Cancer Center between 2003-2021. Patients were stratified into two cohorts: those remaining on active surveillance for ≥ 36 months following diagnosis and those who received within 36 months of diagnosis. Results Between August 2000 and March 2021, we identified 626 patients with a diagnosis of MF. Among these, 48 (8%) did not receive treatment for at least 3 years. Table 1 summarizes the baseline characteristics comparing those pts who remained on active surveillance for ≥ 36 months (LTO-MF) to those who received treatment within 36 months of diagnosis (ET-MF). The LTO cohort presented at a younger age (median age 63 vs 68; p = 0.001), but otherwise demographic variables were balanced between the two cohorts. LTO patients were more likely to have primary MF (85.4% vs 60.9%, p=0.003). LTO patients were less likely to have leukocytosis (28.2% vs 49.9%, p=0.01), and constitutional symptoms (29.8% vs 44.6%, p=0.05), while having a higher reticulocyte percentage (81.4% vs 64.1%, p=0.02). LTO patients also had lower platelet counts (mean: 274k vs 359k, p=0.006), lower percentage of circulating blasts (0.4% vs 1.2%, p<0.001), and lower percentage of marrow myeloblasts (1.3% vs 1.9%, p<0.001) at baseline. Cohorts had comparable rates of anemia, thrombocytopenia, transfusion dependence, LDH levels and splenomegaly at baseline. Interestingly, the cohorts were well-balanced in terms of risk score based across all major prognostic scoring systems: IPSS (p=0.356), DIPSS (p=0.764), DIPSS+ (p=0.148), GIPSS (p=0.125), MIPSS70 (p=0.924) and MIPSS70+ (p=0.407). There was no association between GPSS karyotype risk and need to start treatment earlier (p=0.481) (Table 1). LTO patients were less likely to harbor JAK2 mutations (58.3% vs 72.4%, p=0.04). No significant differences were seen regarding CALR (p=0.144), MPL (p= 0.271), or triple-negative disease (p=0.521) (Table 2). The median OS (mOS) for the entire population was 82.5 months (95%CI 69.4-95.5). LTO patients had longer OS (mOS 170.3 mo vs 63.9 mo; (p<0.001). Rates of transformation to blast phase were comparable (6.2% vs 9.7%;p=0.441), but median time to blast phase transformation was longer for LTO MF: 66.3 mo vs 29 mo, p=0.011). Expectedly, time to first treatment longer for LTO patients (62.1 mo vs 0.9 mo; (p<0.001). No differences were noted between cohorts in terms of response to ruxolitinib, duration of response to ruxolitinib or response to lenalidomide/thalidomide (p = 0.91, 0.90, 0.83, respectively) Conclusion In this single-center study of patients seen at a tertiary referral center, the vast majority of MF patient required treatment within 36 months of diagnosis. Those monitored with active surveillance were younger, had less proliferative signs/symptoms, were less likely to have JAK2 mutations, and more favorable outcomes. Figure 1 Figure 1. Disclosures Tinsley-Vance: Fresenius Kabi: Consultancy; Novartis: Consultancy; Incyte: Consultancy, Speakers Bureau; Abbvie: Honoraria; Jazz: Consultancy, Speakers Bureau; Taiho: Consultancy; Celgene/BMS: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Sallman: Magenta: Consultancy; Takeda: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees. Lancet: AbbVie: Consultancy; BerGenBio: Consultancy; ElevateBio Management: Consultancy; Celgene/BMS: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Agios: Consultancy; Millenium Pharma/Takeda: Consultancy; Jazz: Consultancy. Padron: Incyte: Research Funding; BMS: Research Funding; Taiho: Honoraria; Kura: Research Funding; Blueprint: Honoraria; Stemline: Honoraria. Kuykendall: Novartis: Honoraria, Speakers Bureau; Incyte: Consultancy; BluePrint Medicines: Honoraria, Speakers Bureau; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Research Funding; PharmaEssentia: Honoraria; Abbvie: Honoraria; Celgene/BMS: Honoraria, Speakers Bureau; CTI Biopharma: Honoraria. Komrokji: AbbVie: Consultancy; Geron: Consultancy; Acceleron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees.

Ponatinib in Combination with FLAG-IDA Chemotherapy for Blast-Phase Chronic Myeloid Leukemia: Final Results of the Seamless Phase I/II Dose-Finding UK Trials Acceleration Programme (TAP) Matchpoint Trial

Background Outcomes for patients with blast-phase chronic myeloid leukaemia (BP-CML) are extremely poor, and allogeneic stem cell transplantation (alloSCT) represents the only opportunity for cure. Crucially, long-term survival post-transplant depends on first attaining a return to chronic phase though salvage treatment. Novel strategies that improve response and can optimise transplant outcomes are therefore required. In the era of tyrosine kinase inhibitors (TKIs), BP-CML has become an orphan disease. Consequently, the prospective trials needed to guide clinical practice are rarely attempted. We now report the final results of the prospective MATCHPOINT trial which uses an innovative EffTox design to investigate the activity and tolerability of the TKI ponatinib in combination with high-dose chemotherapy, to improve remission status and transplant outcomes in BP-CML. Methods and patients Between March 2015 and April 2018, 17 patients were recruited through the UK Trials Acceleration Programme to this dose-finding, seamless phase I/II trial of daily ponatinib combined with fludarabine, cytarabine, granulocyte colony-stimulating factor and idarubicin (PON-FLAG-IDA) salvage therapy. We employed EffTox, an advanced Bayesian method to simultaneously consider response to treatment (efficacy) and dose-limiting toxicity (DLT) in all treated patients, providing a single measure of clinical utility, which then informed the subsequent dose level recommendation. The primary objective was to determine the optimal dose of ponatinib, in combination with chemotherapy, as determined by the EffTox model. The primary outcomes were attainment of a second chronic phase and occurrence of a DLT. Secondary outcomes investigated the toxicity of combination therapy, alloSCT outcomes, and survival. The median follow-up of trial patients is 41 months. Results Nine patients completed one cycle of PON-FLAG-IDA, a further eight patients completed both planned cycles. Using an EffTox analysis, the optimal dose of ponatinib was determined as 30mg once daily. Eleven patients achieved a return to chronic phase and four experienced a DLT, fulfilling the pre-specified criteria for clinically relevant efficacy and toxicity. After PON-FLAG-IDA salvage, eight patients attained complete cytogenetic response and five major molecular response (MMR). The most common grade 3-4 non-hematologic toxicities were febrile neutropenia (29% of patients), lung infection (24%), fever (18%) and hypocalcaemia (18%). Three patients experienced treatment-related mortality. Twelve patients proceeded to alloSCT, of whom seven are alive after median 36 months post-transplant follow-up. Only one of the five patients achieving MMR relapsed post-alloSCT, neither of the other relapsing patients achieved a second chronic phase pre-transplant. Median overall survival (OS) of the whole cohort was 12 months (95% confidence interval 6 months to non-calculable), median OS of patients undergoing alloSCT has not been reached. Conclusions Ponatinib has shown that it can be safely combined with high-dose chemotherapy to achieve a return to chronic phase in patients with BP-CML, and represents an effective novel treatment strategy in this high-risk population. Responding patients subsequently undergoing alloSCT can benefit from long-term disease-free survival. The EffTox method enabled very efficient data usage from this high-risk patient population, and is a model for investigating novel therapies in other ultra-orphan cancers. Figure: Overall survival of the MATCHPOINT cohort Figure 1 Figure 1. Disclosures Copland: Incyte: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Jazz: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Cyclacel Ltd: Research Funding. Byrne: Incyte: Honoraria. Rothwell: Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Brock: Eli Lily: Honoraria; Invex Therapeutics: Honoraria; Merck: Honoraria; Roche: Honoraria; AstraZeneca: Current holder of individual stocks in a privately-held company; GSK: Current holder of individual stocks in a privately-held company. De Lavallade: Pfizer, Novartis.: Honoraria; Bristol Myers Squibb, Incyte: Honoraria, Research Funding. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Clark: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria. Smith: Daiichi Sankyo: Speakers Bureau; Pfizer: Speakers Bureau; ARIAD: Honoraria. Milojkovic: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Yap: Faron Pharmaceuticals: Honoraria; Celgene: Honoraria. OffLabel Disclosure: Ponatinib for the treatment of blast-phase CML

Different Treatment Approaches to Blast Phase-Myeloproliferative Neoplasms

CONTEXT: Transformation to acute myeloid leukemia (AML) occurs in 5-20% of patients with myeloproliferative neoplasms (MPN). Overall survival in blast phase MPN (MPN-BP) is poor, usually in the range of 3 to 6 months, and is not significantly impacted by intensive chemotherapy. Current guidelines favor treatment with a hypomethylating agent (HMA), but survival remains poor, and allogeneic hematopoietic stem cell transplantation (AHSCT) holds the only potential for long term survival. OBJECTIVE: To describe the clinical characteristics and overall survival of MPN-BP according to different treatment approaches. DESIGN: Single-institution, retrospective analysis of 70 MPN patients that progressed to blast phase, who presented to our institution between 2001 and 2020. Transformation to AML defined as >20% myeloblasts in peripheral blood or bone marrow. We stratified the patients according to initial treatment strategy for AML. Baseline variables were compared between groups. Median overall survival (mOS) was measured from time of AML diagnosis to date of death. Kaplan-Meier plots were created to compare mOS. RESULTS: Among 70 MF patients that progressed to AML, initial treatment was: 19 best supportive care (BSC), 25 HMA (20 HMA only and 5 HMA + venetoclax), and 26 intensive chemotherapy (IC) [12 patients received standard "7+3" regimen with daunorubicin/idarubicin and cytarabine, 12 received high-dose cytarabine, cladribine +/- mitoxantrone (CLAG/CLAG-M), and 2 received CPX-351 (Vyxeos)]. Patients receiving IC were younger at time of leukemic transformation than those receiving BSC (median 63.9 years vs 72.9 years; p=0.029) or HMA (median 63.9 years vs. 69.0 years; p=0.026). Additionally, 70% of IC patients had an ECOG performance status of 0 or 1 compared to just 48% of patients receiving either BSC or HMA (p=0.088). Median OS for the entire cohort (n = 70) was 4.8 months. Compared to patients who received active treatment with HMA or IC, those treated with BSC had shorter survival (0.9 months vs 6.4 months; p=0.001). Median survival between patients treated with HMA and IC was not significantly different (4.5 months vs 9.6 months; p=0.13). Patients treated with IC were more likely to proceed to AHSCT (46% vs 5%; p < 0.001). Between HMA and IC groups, there was no difference in time from MPN-BP diagnosis to treatment (median 0.4 months vs 0.3 months; p=0.644) or total number of lines of treatment for MPN-BP. Focusing specifically on the role of AHSCT in patients treated with IC, we found that patients who received AHSCT had significantly longer mOS than those patients who did not (18.9 months vs 4.9 months; p=0.002), suggesting the beneficial role of intensive chemotherapy is critically tied to the ability to subsequently undergo AHSCT. Among patients who underwent AHSCT, 1-year and 2-year OS was 51% and 34%, respectively. In contrast, patients not receiving AHSCT had 1-year and 2-year OS of 14% and 2%, respectively. Independent of age, AHSCT (p=0.008) and receipt of therapy (p=0.017) significantly correlated with longer survival after AML diagnosis. Besides these factors, there were no significant differences in the clinical characteristics between the three groups. Acknowledging the limitations associated with small numbers, we did not note any difference in survival between patients who received HMA vs HMA + venetoclax (p=0.27). CONCLUSIONS: In MPN-BP, patients receiving treatment had superior outcomes to those that received BSC. Initial treatment with intensive chemotherapy was associated with non-significant improvement in survival; however, this appears to be critically linked to the receipt of AHSCT. In appropriate patients, intensive chemotherapy may be reasonable in an effort to provide an effective bridge to AHSCT. Still, this study reinforces the poor prognosis associated with MPN-BP and the desperate need for novel therapeutic approaches in this group of patients. Figure 1 Figure 1. Disclosures Sallman: AbbVie: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Kite: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau. Sweet: Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Padron: BMS: Research Funding; Kura: Research Funding; Incyte: Research Funding; Blueprint: Honoraria; Taiho: Honoraria; Stemline: Honoraria. Lancet: Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Millenium Pharma/Takeda: Consultancy; BerGenBio: Consultancy; AbbVie: Consultancy; Astellas: Consultancy; Agios: Consultancy; ElevateBio Management: Consultancy; Jazz: Consultancy. Komrokji: PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; Acceleron: Consultancy; Jazz: Consultancy, Speakers Bureau; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kuykendall: Novartis: Honoraria, Speakers Bureau; Prelude: Research Funding; Incyte: Consultancy; PharmaEssentia: Honoraria; CTI Biopharma: Honoraria; Celgene/BMS: Honoraria, Speakers Bureau; BluePrint Medicines: Honoraria, Speakers Bureau; Abbvie: Honoraria; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Custom High Throughput Drug Sensitivity Assay Reveals Therapeutic Options for Chronic Myeloid Leukemia Patients Resistant to or Intolerant of Tyrosine Kinase Inhibitors

Introduction. Tyrosine kinase inhibitors (TKIs) have revolutionized chronic phase (CP) chronic myeloid leukemia (CML) care with many patients achieving major and deeper molecular responses. However, for those who are resistant to or do not tolerate the approved TKIs, there are few alternatives. We therefore developed a custom high throughput drug screen comprised of both FDA approved and investigational agents. Methods. Fifty-six samples (50 individual patients) have undergone testing in the drug sensitivity assay, for which a large fraction exhibited resistance to approved agents. The Quellos High Throughput Core Laboratory's Cancer Drug Sensitivity has been CLIA approved for leukemia since 2015. Blood and bone marrow samples were obtained from CML patients with written informed consent. Mononuclear cells were isolated by density depletion. The myeloid population was obtained by lineage depletion of non-myeloid cells using magnetic beads and antibodies to erythroid lineage (CD235a), T (CD3) and B (CD19) lymphocytes, and NK (CD56) cells. Flow cytometry confirmed successful enrichment of the myeloid cell population. Cells were plated on extracellular matrix coated 384 well plates to test under conditions of adhesion mediated chemotherapy resistance. Initially, the assay was comprised of 32 drugs (11 patients) selected based on published activity in CML and resistant CML. The assay was then expanded to 64 drugs. Compounds are added (ranging from 5 pM to 100 μM) to patient samples using the CyBio CyBi-Well Vario and incubated at 37°C, 5% CO2 for 72 hours, then viability is assessed by CellTiterGlo. IC50s and AUCs are calculated for each drug using XLFit (IDBS) and a standard 4 parameter logistical model. Transcriptome analysis is planned for these samples. Results. Clinical characteristics are shown in Table 1. Mean and median BCR-ABL1 transcripts were 69% and 70% in diagnosis samples and 63% and 55% in resistant samples, respectively (P=0.607). ABL mutations were present in 5 patients (M244V, T315I, F359I). Additional myeloid mutations were present in 5 of 6 evaluable advanced phase samples, 4 of 17 evaluable diagnostic samples, and 3 of 10 evaluable resistant samples and included ASXL1, DNMT3A, IDH1, JAK2V617F, NRAS, RUNX1, and TET2. Figure 1 illustrates the breadth of sensitivity to agents in the assay. Figure 2 is a heat map of area under the curve (AUCs) illustrating the unique drug sensitivity patterns for all patients, with unsupervised clustering. For new diagnosis patients, the TKIs imatinib, dasatinib, nilotinib, bosutinib, and ponatinib ranked in the top 8 drugs. For primary resistant patients, the IC50 values for imatinib, nilotinib, bosutinib, and ponatinib were higher than the new diagnosis patients. For example, for ponatinib, the mean IC50 was 402.6 ± 354.7 X 10 E-9 M for primary resistant samples vs. 1.65 ± 0.45 X 10 E-9 M for diagnosis group, p=0.015 (Welch t test), or about 250-fold higher (less sensitive). In accelerated and blast phase samples drugs with IC50 values lower than 0.1 µM, a range that could correlate with in vivo drug response, were identified for all patients (range, 3-20 drugs per patient). Top candidates included proteasome and kinase inhibitors. In 2 patients harboring NRAS mutations, IC50 for trametinib was less than 0.1 µM as compared to patients without NRAS mutations, where the IC50s were higher. Clinical outcomes are available for nearly all patients. Although the study was not designed to select next-line TKI therapy in resistant patients, drug profiling was informative in many cases. Data for 7 resistant patients are shown in Table 2. For example, CML-012 had the lowest IC50 value (indicating most sensitive) for dasatinib, 4.1 X 10E-8 M and responded to dasatinib after failing imatinib (IC50 8.4 X 10E-7M). CML-003 did not respond to bosutinib (IC50 1.2 X 10E-6M) and did respond to dasatinib (IC50 1.2 X 10E-7M). CML-056 did not respond to nilotinib (IC50 1.4 X 10E-6M), dasatinib (IC50 6.9 X 10E-4M), or ponatinib (IC50 1.0 X 10E-6M). Notably, in all resistant patient samples we identified drugs with IC50 values lower than 0.1 µM. These therapeutics can be prioritized for further evaluation, either alone or in combination with TKIs, in resistant CML patients. Conclusion. In vitro drug sensitivity testing provides data for potential agents for patients with resistance or intolerance to FDA approved TKIs, or those that have entered accelerated phase or blast phase. Figure 1 Figure 1. Disclosures Oehler: Blueprint Medicines: Consultancy; Takeda: Consultancy; Pfizer: Research Funding; OncLive: Honoraria; BMS: Consultancy. Becker: Abbie: Research Funding; SecuraBio: Research Funding; Cardiff Oncology: Research Funding; Pfizer: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Glycomimetics: Research Funding. OffLabel Disclosure: We developed a custom high throughput drug screen comprised of both FDA approved and investigational agents

Navtemadlin (KRT-232), a Small Molecule MDM2 Inhibitor, Is More Effective Than Decitabine Against Myeloproliferative Neoplasm-Blast Phase in a Patient-Derived Xenograft Model

Patients with myeloproliferative neoplasm-blast phase (MPN-BP) have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies (Mesa Blood 2015). Decitabine is the standard of care for MPN-BP. Recently, using xenotransplantation assays, we have shown that MPN-BP originates at the hematopoietic stem cell (SC) level (Wang Blood 2018) and that MPN-BP CD34 + cells contain higher levels of MDM2 protein compared with their normal counterparts (% of MDM2 + CD34 + cells: MPN-BP: 76.4±3.3; Normal: 17.5±6.4. P <0.05). MDM2 negatively regulates p53 activity and MDM2 inhibitors can activate p53 and induce apoptosis of TP53 WT cancer cells. As mutations or deletions of TP53 occur infrequently in MPN-BP, we examined the effects of a potent MDM2 inhibitor, navtemadlin (KRT-232; Canon Mol. Cancer Ther. 2015) and decitabine as monotherapy or in combination on the depletion or elimination of MPN-BP cells in a patient derived xenograft (PDX) model. We firstestablished the dynamics of leukemia cell recovery following a single cycle of navtemadlin treatment. Spleen cells with wild type (WT) TP53 and mutations in KRAS, RAD21, KMT2A and ASXL1 were collected from the 4 th generation of MPN-BP PDX mice. Forty days after injection of these spleen cells (4×10 5/mouse) into sublethally irradiated NSG mice, peripheral blood (PB) leukemic burden (hCD34 + cells: 0.39±0.09%) was demonstrated by flow cytometric analysis. Mice were treated with vehicle alone (n=3) or high doses of navtemadlin (100 mg/kg, n=4) by daily oral gavage on day (D) 1-7. Without navtemadlin treatment, leukemia engraftment and leukemic burden continued to increase until the mice died on D15-D23. By contrast, hCD34 + leukemic blasts were almost undetectable on D8 and remained at significantly lower levels in PB of mice treated with navtemadlin on D15, than were detected in mice receiving vehicle alone (D8: Vehicle: 1.8±1.3%; Navtemadlin: 0.1±0.1%. D15: Vehicle: 19.9±6.0%; Navtemadlin: 0.5±0.1%). These findings suggest that navtemadlin monotherapy has the potential to deplete MPN-BP blast cells and prolong survival in MPN-BP PDX mice. To achieve long-term remission and to prevent relapse, we treated MPN-BP PDX mice with multiple cycles of low (50 mg/kg, n=4) and high dose navtemadlin (100 mg/kg, n=5) at three-week intervals based on the dynamics of leukemia cell recovery following a single cycle of navtemadlin treatment. hCD34 + cells, which contain MPN-BP SCs, and hCD45 dimCD33 + leukemic blasts were reduced in the spleen, but not in the marrows of mice during 3 cycles of 100 mg/kg navtemadlin treatment. However, reduced leukemia cell burden in PB persisted during 3 cycles of navtemadlin treatment and was associated with a prolongation in survival, which was dose-dependent (Mean survival after transplantation: Vehicle: 64.0 days; 50mg/kg navtemadlin: 86.0 days; 100mg/kg navtemadlin: 98.3 days). We then examined if a combination of navtemadlin and decitabine is more effective than single agent therapy in depleting MPN-BP SCs. Again, navtemadlin at 100 mg/kg significantly reduced the leukemia cell burden in mouse PB on C1D8 (hCD34 + cells: Vehicle: 4.1±0.8%; Navtemadlin: 0.6±0.2%, an 85% decrease. hCD45 dimCD33 + cells: Vehicle: 1.4±0.3%; Navtemadlin: 0.04±0.03%, a 97% decrease. P <0.001 for both), which persisted during 2 cycles of treatment. By contrast, multiple cycles of decitabine monotherapy (2.5mg/kg, IP, 3 times/week in 21-day cycles) resulted in a modest reduction in hCD34 + cells and hCD45 dimCD33 + cells (11.4% and 30.1% decrease, respectively) in PB of MPN-BP PDX mice, and did not reduce these cells in either the spleen or bone marrow on C1D8. Finally, addition of decitabine to navtemadlin did not further deplete either MPN-BP SCs or leukemia cells and did not improve survival of MPN-BP PDX mice, as compared to treatment with navtemadlin alone. Mean survival after transplantation in the combination study was: Vehicle: 55.5 days; navtemadlin: 70.3 days; Decitabine: 56.3 days; Combination: 64.0 days. Furthermore, toxicity (body weight loss, intestinal pathology) was observed in mice receiving high dose of navtemadlin and decitabine simultaneously, but not when either drug was administered alone. In conclusion, navtemadlin monotherapy, which activates p53, depletes leukemia cell counts and prolongs survival of MPN-BP PDX mice and is a promising agent for patients with WT TP53 MPN-BP. Disclosures Krejsa: Kartos Therapeutics, Inc.: Current Employment. Hoffman: AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding.

Efficacy of HMA +/- Venetoclax or Intensive Chemotherapy in Blast-Phase Myeloproliferative Neoplasms

Introduction Blast-phase (BP), or leukemic transformation is a rare and devastating complication of myeloproliferative neoplasms (MPNs) (primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocythemia (ET), and post-PV/ET myelofibrosis). Patients with BP-MPNs have a poor prognosis with a median overall survival of less than 6 months, and there is no standard treatment regimen for these aggressive diseases (Dunbar et al, Blood. 2020). The development of hypomethylating agent (HMA)/venetoclax (Ven) combination offers new hope for some with AML, but has been relatively disappointing in BP-MPN, though data are limited and retrospective (Masarova et al, Blood Adv. 2021; Gangat et al, Am J Hematol. 2021). Here, we add our experience with several common treatment regimens for BP-MPN. Methods We retrospectively analyzed data from 39 consecutive patients with BP-MPNs diagnosed from December 2008 to February 2021 who received treatment at the University of Michigan. We included all patients with a previous diagnosis of MPNs who had ≥ 20% blasts in the peripheral blood or bone marrow, and subsequently received systemic therapy. One patient with a myeloid sarcoma was included as well. Disease characteristics at time of BP-transformation were noted. Patients were divided into the following groups based on 1 st-line induction therapy: 7+3 (daunorubicin and cytarabine), FLAG (fludarabine, high-dose cytarabine and G-CSF), hypomethylating agent only (decitabine or azacitidine), and HMA/Ven. Patients were followed for 2 years post-diagnosis. Differences in induction response were assessed using the Chi-square test. Differences in overall survival were calculated using the Kaplan-Meier regression with the log-rank test. Two patients who received alternate induction therapy outside of the four groups were not included in these analyses. Results The composite BP-MPN population had a median advanced age of 69 years old and a median ECOG performance status (PS) of 1. Most (97.4%) had received systemic treatment prior to their transformation for their MPN, with 71.8% receiving hydroxyurea and 41.0% receiving ruxolitinib. The rate of response (CR, CRi, MLFS) was highest in the HMA/Ven group at 42.9%, followed by FLAG (29.4%), HMA only (11.1%), and 7+3 (0%), p = 0.033 (Table 3). Despite the higher response rate, differences in 2-year OS were not significantly different among the 4 groups: 7+3 (25.0%), FLAG (7.7%), HMA (0%), HMA/Ven (20.0%) (p=0.92, Figure 1). Median time to relapse after achieving remission ranged from 2-10 months, and did not vary significantly based on induction regimen. Patients in the HMA and HMA/Ven groups had higher incidences of death with induction at 77.8% and 28.6%, respectively (Table 3). Conclusions The highest rates of response, including complete remission, were achieved with the combination of HMA and venetoclax compared to intensive induction chemotherapy or HMA alone. However, this did not translate into significant differences in OS, which is consistent with other retrospective reports. No responses were seen with 7+3 induction, though several patients were able to go on salvaged with other therapies and subsequent allogeneic stem cell transplantation thereafter. Finally, the baseline poor ECOG PS of the HMA group and borderline ECOG PS of the HMA/Ven group also contribute to their low survival rates.Larger, prospective studies comparing currently available treatment regimens in BP-MPN would be helpful, but ultimately new therapies are desperately needed for this high-risk disease. Figure 1 Figure 1. Disclosures Bixby: Takeda: Consultancy. Talpaz: Constellation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Grant/research support ; Celgene: Consultancy; Imago: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.