Patnaik, Mrinal M.

Background: Recent advances in the treatment of myelodysplastic syndrome (MDS) have improved patient survival and quality of life (QOL), while reducing transfusion burden. However, allogeneic hematopoietic cell transplantation (HCT), widely used in younger MDS patients, remains the only curative therapy for MDS. While transplantation outcomes among selected older patients with MDS are similar to younger patients with MDS, early transplantation for older patients is infrequently offered since the relative benefits of HCT over non-HCT therapy in have not been well defined in this patient group. We conducted a multi-center, biologic assignment trial in older individuals with high risk MDS to define the benefit of HCT over non-HCT therapy. Methods: The study was a multicenter, biologic assignment trial in subjects aged 50-75 with higher risk de novo MDS (IPSS Intermediate-2 (Int-2) or High) who were candidates for reduced-intensity conditioning (RIC) allogeneic HCT, comparing outcomes of those with a suitable 8/8 HLA-matched donor to those without a donor. The trial was conducted by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN 1102, NCT02016781). Eligible subjects were enrolled prior to a formal donor search, and before or after MDS treatment was initiated. Biological assignment to the Donor or No Donor arm was based on high-resolution HLA typing of eligible family members and a search of the unrelated donor registries. Subjects were initially assigned to the No Donor arm and re-assigned to the Donor arm when a suitable donor was identified. Subjects who died or whose 90-day donor search ended without identifying a suitable donor remained in the No Donor arm. Subjects in the Donor arm were expected to undergo RIC HCT within 6 months of enrollment. Subjects underwent RIC HCT or non-HCT therapy according to institutional standards. The primary analysis compared three-year overall survival (OS) between arms using adjusted survival estimates to account for the potential bias resulting from biological assignment. The sample size was selected to provide at least 80% power to detect a difference of 15% in 3-year OS. Between January 2014 and November 2018, 384 subjects (Donor n=260, No Donor n=124) were enrolled at 34 centers. The study arms were well balanced for age, gender, KPS, IPSS risk, MDS disease duration and responsiveness to hypomethylating therapy (Table). The median follow-up time for surviving patients was 34.2 months (range: 2.3-38 months) in the Donor arm and 26.9 months (range: 2.4-37.2 months) in the No Donor arm. Results: In an intent-to-treat analysis, adjusted OS at 3 years from study enrollment in the Donor arm was 47.9% (95% CI: 41.3%-54.1%) compared with 26.6% (95% CI: 18.4%-35.6%) in the No Donor arm (p=0.0001, absolute difference 21.3%, 95% CI: 10.2%-31.8%)(Figure). A sensitivity analysis excluding subjects assigned to the No Donor arm who died or withdrew prior to the end of the 90-day search window showed no effect on outcome (Adjusted OS: 48.0% vs. 28.1%, p=0.0004). Leukemia-free survival (LFS) at 3 years was greater in the Donor arm (35.8%, 95% CI: 29.8%-41.8%) compared with the No Donor arm (20.6%, 95% CI: 13.3%-29.1%, p=0.003), with no changes in the sensitivity analysis. An OS and LFS benefit was seen across all subgroups tested (Figure). There were no clinically significant differences in QOL between Donor and No Donor arms as measured by the FACT-G, the MOS-SF36 Physical and Mental Component Scores and the EQ-5D utility score at all time points. The overall non-compliance rate for the trial was 26.3%. Reasons for non-compliance included the use of myeloablative conditioning or failure to proceed to RIC transplant in the Donor arm, and the use of alternative donors in the No Donor arm. In an as-treated analysis, comparison of the HCT and No HCT arms demonstrated a significant advantage in 3-year OS (47.4% vs. 16.0%, p<0.0001) and LFS (39.3% vs. 10.9%, p<0.0001) for subjects who underwent HCT. Conclusions: We observed a significant OS advantage in older patients with Int-2 and High IPSS risk de novo MDS who are RIC HCT candidates and have an HLA-matched donor, when compared with those without a donor. The benefit of having a matched donor was seen across subgroups, including those who were of Medicare age (>65) and below. HCT should be offered to all individuals between the ages of 50-75 with Int-2 and High IPSS risk MDS in whom a suitable donor can be identified. Table Disclosures Nakamura: Magenta Therapeutics: Other: Advisory board meeting; Kyowa-Kirin: Other: Support on a meeting presentation; Alexion: Other: Support on a meeting presentation; Merck: Other: advisory board meeting; NapaJen Pharma: Consultancy; Kadmon Corporation: Other: Advisory board meeting; Celgene: Other: Support on seminar; Viracor: Consultancy. Scott:Alexion, Incyte, Novartis, Regeneron: Consultancy; Agios, BMS: Honoraria; BMS, Novartis: Research Funding. Oran:ASTEX: Research Funding; Celgene: Consultancy; Arog Pharmaceuticals: Research Funding. Maziarz:Novartis, Incyte, CRISPR Therapeutics, Artiva Biotherapeutics, and AlloVir: Consultancy; Novartis and Athersys: Other: DSMB participant; Athersys: Patents & Royalties; Incyte, Kite, BMS/Celgene, PACT Pharma, Orca BioSystems, and Omeros: Honoraria; Novartis and Juno: Research Funding. McGuirk:Allo Vir: Consultancy, Honoraria, Research Funding; Gamida Cell: Research Funding; Pluristem Ltd: Research Funding; Bellicum Pharmaceutical: Research Funding; Astellas: Research Funding; Fresenius Biotech: Research Funding; Novartis: Research Funding; Kite Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Juno Therapeutics: Consultancy, Honoraria, Research Funding. Sekeres:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 32,270 in 2020, from SEER). Despite recent treatment advances, patients diagnosed with high risk/refractory MM (hrrMM) have an overall survival of only three years. Recently, our lab developed the first genetically engineered NrasQ61R; Myc-driven myeloma model (VQ mice). The VQ model recapitulates most biological and clinical features of hrrMM. VQ cells are serially transplantable into syngeneic recipients and can be propagated in vitro. As such, the VQ model could serve as a robust pre-clinical platform for drug screening and in vivo testing of new therapies to treat hrrMM. Whole exome sequencing (WES) of CD138+ cells from five primary VQ mice identified eight recurrently mutated genes. Of these eight genes, two have been previously identified as recurrently mutated in MM patients while the remainder appear to be unique to VQ myeloma. Meanwhile, RNA sequencing of CD138+ cells taken from both primary VQ mice and secondary VQ recipients show differential gene expression for five of these eight genes. Gene ontology analysis of recurrently mutated genes indicates that these genes play a role in interferon signaling, as well as receptor tyrosine kinase signaling. Here, we report the results of a high-throughput repurposing screen using the NCI's AOD IX library, which consists of 147 FDA-approved anti-cancer drugs. VQ cells were treated with 0.1 and 1µM of each compound in the presence or absence of 10nM of Trametinib (Tra), a potent MEK inhibitor. Screening results returned expected positive hits that were previously FDA approved for MM treatment (green dots, Panel 1B), such as proteasome inhibitors, histone deacetylase inhibitors, and chemotherapy drugs. Importantly, VQ cells were resistant to some compounds currently used to treat MM, including the BCL-2 inhibitor Venetoclax, the alkylating agent Melphalan, and the topoisomerase II inhibitor Etoposide (red dots, Panel 1B). De novo resistance to these therapies further indicates that the VQ cells model a multi-drug resistant MM. Two compounds were chosen for further testing: the protein translation inhibitor Omacetaxine (Oma) based on its single agent efficacy, and the multi-receptor tyrosine kinase inhibitor Ponatinib (Pot) based on its high synergy with Tra. Combination Index testing confirmed initial drug screening results but showed some antagonism between combination Oma and Pot treatment. We proceeded with in vivo testing of two novel combination therapies, in which 3-week treatment cycles were followed by a 1-week rest period between cycles: Three weeks of daily Tra and Pot treatment (TP), or two weeks of Tra and Pot treatment followed by one week of Oma (TPO) treatment. Vehicle (Veh) treated and Tra only treated mice were used as controls. Treatment efficacy was assessed against VQ-D1 MM, which grows primarily in the bone marrow (BM) and metastasizes to the spleen and liver of recipient mice. Four weeks post-treatment, myeloma growth in the BM was significantly reduced in both combo treatment groups, while MM burden in the spleen was also reduced. Overall, both TP and TPO treatment significantly extended survival compared to Veh or Tra treated mice, and significantly reduced liver extramedullary disease. To further test the range of Tra/Pot synergy in vivo, Tra dosage was reduced and treatment was administered daily. Interestingly, both low dose Tra monotherapy and TP combo therapy significantly extended survival compared to Veh control or Pot alone, and TP treated mice had increased survival compared to the Tra only treated mice. Taken together, our results show that low dose daily Trametinib treatment is superior to cycling dosage, and that Ponatinib treatment synergizes with both dosing schedules to extend survival in vivo. Our study demonstrates the utility of the VQ model for identifying new treatments for hrrMM and testing their efficacy in vivo. As part of our ongoing drug repurposing experiments, we have expanded our screening to a panel of 2,580 FDA-approved drugs, including but not limited to anti-cancer and anti-inflammatory drugs. Figure Disclosures Callander: Cellectar: Research Funding; University of Wisconsin: Current Employment.

Abstract Objectives SF3B1 mutations are the most common mutations in myelodysplastic syndromes (MDS). The International Working Group for the Prognosis of MDS (IWG-PM) recently proposed SF3B1-mutant MDS (SF3B1-mut-MDS) as a distinct disease subtype. We evaluated the spectrum and molecular landscape of SF3B1-mutated myeloid disorders and assessed the prognostication in MDS harboring SF3B1 mutations (MDS-SF3B1). Methods Cases were selected by retrospective review. Clinical course and laboratory and clinical findings were collected by chart review. SF3B1-mut-MDS was classified following IWG-PM criteria. Results SF3B1 mutations were identified in 75 of 955 patients, encompassing a full spectrum of myeloid disorders. In MDS-SF3B1, Revised International Prognostic Scoring System (IPSS-R) score greater than 3 and transcription factor (TF) comutations were adverse prognostic markers by both univariate and multivariate analyses. We confirmed the favorable outcome of IWG-PM-defined SF3B1-mut-MDS. Interestingly, it did not show sharp prognostic differentiation within MDS-SF3B1. Conclusions SF3B1 mutations occur in the full spectrum of myeloid disorders. We independently validated the favorable prognostication of IWG-PM-defined SF3B1-mut-MDS. However it may not provide sharp prognostication within MDS-SF3B1 where IPSS-R and TF comutations were prognostic-informative. Larger cohort studies are warranted to verify these findings and refine MDS-SF3B1 prognostication.

AbstractDisease OverviewRing sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include myelodysplastic syndromes with RS (MDS‐RS) and MDS/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN‐RS‐T).DiagnosisMDS‐RS is a lower risk MDS, with single or multilineage dysplasia (MDS‐RS‐SLD/MLD), <5% bone marrow (BM) blasts, <1% peripheral blood blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN‐RS‐T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS‐RS‐SLD, along with a platelet count ≥450 × 109/L and large atypical megakaryocytes.Mutations and KaryotypeMutations in SF3B1 are seen in ≥80% of patients with MDS‐RS‐SLD and MDS/MPN‐RS‐T, and strongly correlate with the presence of BM RS; MDS/MPN‐RS‐T patients also demonstrate JAK2V617F (50%), DNMT3A, TET2 and ASXL1 mutations. Cytogenetic abnormalities are uncommon in both.Risk StratificationMost patients with MDS‐RS‐SLD are stratified into lower risk groups by the revised‐IPSS. Disease outcome in MDS/MPN‐RS‐T is better than that of MDS‐RS‐SLD, but worse than that of essential thrombocythemia (MPN). Both diseases are associated with a low risk of leukemic transformation.TreatmentAnemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Luspatercept, a first‐in‐class erythroid maturation agent is now approved for the management of anemia in patients with MDS‐RS and MDS/MPN‐RS‐T. Aspirin therapy is reasonable in MDS/MPN‐RS‐T, especially in the presence of JAK2V617F, but the value of platelet‐lowering drugs remains to be defined.

Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

AbstractMyelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are unique myeloid neoplasms, with overlapping features of MDS and MPN. They consist of four adult onset entities including chronic myelomonocytic leukemia (CMML), MDS/MPN-ring sideroblasts-thrombocytosis (MDS/MPN-RS-T), BCR-ABL1 negative atypical chronic myeloid leukemia (aCML) and MDS/MPN-unclassifiable (MDS/MPN-U); with juvenile myelomonocytic leukemia (JMML) being the only pediatric onset entity. Among these overlap neoplasms, CMML is the most frequent and is hallmarked by the presence of sustained peripheral blood monocytosis with recurrent mutations involving TET2 (60%), SRSF2 (50%) and ASXL1 (40%); with RAS pathway mutations and JAK2V617F being relatively enriched in proliferative CMML subtypes (WBC ≥13 × 109/L). CMML usually presents in the 7th decade of life, with a male preponderance and is associated with a median overall survival of &lt;36 months. Adverse prognosticators in CMML include increasing age, high WBC, presence of circulating immature myeloid cells, anemia, thrombocytopenia and truncating ASXL1 mutations. While allogeneic stem cell transplantation remains the only curative option, given the late onset of this neoplasm and high frequency of comorbidities, most patients remain ineligible. Hypomethylating agents such as azacitidine, decitabine and oral decitabine/cedazuridine have been US FDA approved for the management of CMML, with overall response rates of 40-50% and complete remission rates of &lt;20%. While these agents epigenetically restore hematopoiesis in a subset of responding patients, they do not impact mutational allele burdens and eventual disease progression to AML remains inevitable. Newer treatment modalities exploiting epigenetic, signaling and splicing abnormalities commonly seen in CMML are much needed.

Abstract Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are uniquely classified neoplasms occurring in both children and adults. This category consists of 5 neoplastic subtypes: chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), BCR-ABL1–negative atypical chronic myeloid leukemia (aCML), MDS/MPN-ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN-unclassifiable (U). Cytogenetic abnormalities and somatic copy number variations are uncommon; however, &gt;90% patients harbor gene mutations. Although no single gene mutation is specific to a disease subtype, certain mutational signatures in the context of appropriate clinical and morphological features can be used to establish a diagnosis. In CMML, mutated coexpression of TET2 and SRSF2 results in clonal hematopoiesis skewed toward monocytosis, and the ensuing acquisition of driver mutations including ASXL1, NRAS, and CBL results in overt disease. MDS/MPN-RS-T demonstrates features of SF3B1-mutant MDS with ring sideroblasts (MDS-RS), with the development of thrombocytosis secondary to the acquisition of signaling mutations, most commonly JAK2V617F. JMML, the only pediatric entity, is a bona fide RASopathy, with germline and somatic mutations occurring in the oncogenic RAS pathway giving rise to disease. BCR-ABL1–negative aCML is characterized by dysplastic neutrophilia and is enriched in SETBP1 and ETNK1 mutations, whereas MDS/MPN-U is the least defined and lacks a characteristic mutational signature. Molecular profiling also provides prognostic information, with truncating ASXL1 mutations being universally detrimental and germline CBL mutations in JMML showing spontaneous regression. Sequencing information in certain cases can help identify potential targeted therapies (IDH1, IDH2, and splicing mutations) and should be a mainstay in the diagnosis and management of these neoplasms.

PURPOSE Patients with lower-risk (LR) myelodysplastic syndromes (MDS) who are RBC transfusion dependent and have experienced relapse after or are refractory to erythropoiesis-stimulating agent (ESA) have limited treatment options. High telomerase activity and human telomerase reverse-transcription expression in clonal hematopoietic cells have been reported in patients with MDS. Imetelstat, a first-in-class competitive inhibitor of telomerase enzymatic activity, targets cells with active telomerase. We report efficacy, safety, and biomarker data for patients with LR MDS who are RBC transfusion dependent and who were relapsed/refractory to ESAs. PATIENTS AND METHODS In this two-part phase II/III study (MDS3001), the primary end point was 8-week RBC transfusion independence (TI) rate, with key secondary end points of 24-week RBC TI rate, TI duration, and hematologic improvement-erythroid. RESULTS Data from the phase II part of the study are reported. Of 57 patients enrolled and treated (overall population), 38 were non-del(5q) and hypomethylating agent and lenalidomide naïve (subset population). The 8- and 24-week RBC TI rates in the overall population were 37% and 23%, respectively, with a median TI duration of 65 weeks. In the subset population, 8- and 24-week RBC TI rates were 42% and 29%, respectively, with a median TI duration of 86 weeks. Eight-week TI rate was observed across all subgroups evaluated. Cytogenetic and mutational data revealed a reduction of the malignant clones, suggesting disease modification activity. The most common adverse events were cytopenias, typically reversible within 4 weeks. CONCLUSION Imetelstat treatment results in a meaningful, durable TI rate across a broad range of heavily transfused patients with LR MDS who are ineligible for or relapsed/refractory to ESAs. Biomarker analyses indicated effects on the mutant malignant clone.

Introduction: Recent evidence has demonstrated that Clonal Hematopoiesis of Indeterminate Potential (CHIP) increases the risk of cardiovascular events. However, the exact role played by CHIP in early stages of coronary atherosclerosis, characterized by coronary endothelial dysfunction remains to be elucidated. Hypothesis: The current study was designed to test the hypothesis that the presence of CHIP in peripheral blood cells is associated with coronary endothelial dysfunction and increased levels of inflammatory markers. Methods: Next generation sequencing was used to detect mutations among patients who had coronary endothelial dysfunction and control group (normal coronary endothelial function). Endothelial dysfunction was defined by ≥ 20% decrease in coronary artery diameter or ≤50% increase in blood flow in response to acetylcholine injection compared to baseline. Plasma cytokine levels of Interleukin (IL)-6 and IL-8 were also assessed. Results: Clonal hematopoiesis relevant gene mutations were found in 1 out of 64 patients with normal endothelial function (1.5%) and 11 out of 119 cases in the endothelial dysfunction group (9.2%) (P = 0.04) with ASXL1 (Additional sex combs-like 1) being the most frequent gene (4.2%) mutated. Cytokine analysis demonstrated that mutations in ASXL1, DNMT3A (DNA methyltransferase 3A) and TET2 (Ten-eleven-translocation-2) in the endothelial dysfunction group were associated with increased levels of IL-6 and IL-8 (P = 0.001, P = 0.003; respectively). Conclusions: The current study supports a potential role for CHIP in mechanisms of coronary artery disease starting at early stage of atherosclerosis. Furthermore, enhanced expression levels of IL-6 and IL-8 seem to be related to mutations in DNMT3A, ASXL1 and TET2 genes, more than other gene mutations relevant CHIP.