Patnaik, Mrinal M.

Key Points Systemic immune microenvironment signatures in CMML indicate an altered T- and natural killer cell balance. CMML bone marrow dendritic cell aggregates associate with disease progression and systemic regulatory T-cell phenotypic switch.

Introduction: Mutations in the gene isocitrate dehydrogenase 2 (IDH2) occur in ~12-15% of patients with acute myeloid leukemia (AML). Enasidenib, an oral small molecule selective targeted inhibitor of IDH2 enzymes, was approved forIDH2mutated relapsed/refractory AML (RR-AML) in 2017 (Stein et. al., 2017). There is limited data regarding its efficacy and toxicity outside the context of clinical trials. Using Mayo Clinic's cohort of AML patients, we provide a real-world look at outcomes forIDH2mutated RR-AML patients treated with enasidenib. Methods: A total of 13 patients treated with enasidenib for RR-AML were identified, all of which experienced at least one relapse following induction chemotherapy. Response assessment was based on European Leukemia Net (ELN) 2017 response criteria.Complete blood counts including blast percentage and transfusion dependency were obtained at AML diagnosis, initiation of enasidenib, 2 months and 6 months following initiation of therapy. Transfusion dependency was defined by the need for either a red cell or platelet transfusion within 4 weeks of the designated time frame. We compared the clinical characteristics of patients who achieved complete response (CR) (n=4) to those not achieving CR (n=9), using the Mann-Whitney U test for numerical variables and the Fischer's Exact test for quantitative data. Results: The median age at diagnosis of our 13 patients was 68 years (range 60-82), with a male predilection (n=11) (Table 1). Cytogenetic and molecular genetic findings are provided in Table 2. Complete response (CR) was achieved in 4 (30.7%) patients by cycle 2 and 2 of those patients (50%) proceeded to an allogeneic hematopoietic stem cell transplant (AHSCT). The median time from diagnosis to initiation of treatment for patients in CR was 3.5 months (range 1-7), and for patients not achieving CR was 13 months (range 6-53; p=0.048), with median time of follow up after treatment initiation of 23.5 months (range, 5-36) and 7 months (range, 1-14), respectively. Among 8 patients who were transfusion dependent at baseline, all the CR patients (2/2) achieved red blood cell and platelet transfusion independence 6 months after treatment, instead only (3/6) 50% of those not in CR achieved transfusion independence (p=0.028). An initial trajectory of improvement in counts is noted from the time of diagnosis to the 2 month mark post treatment with 75% of the responder cohort showing improvement in counts which was durable through the 6 month mark, whereas 55% of those that did not respond had worsening counts at month 2 and died prior to 6 month follow up. At the time of data cut-off, the median duration of CR was 14 months (range, 2-33), with 3 of 4 (75%) of responders maintaining CR. Figure 1 details overall responses and outcomes in all 13 patients. None of the patients in our study experienced differentiation syndrome. Five patients were hospitalized in the course of treatment for infectious complications. Two patients were noted to have hyperbilirubinemia however there was no clear indication that this was secondary to enasidenib Median overall survival for all 13 patients was 21 months (range, 6-53 months) with leukemia free survival of 7 months (range, 5-19 months). Furthermore, median overall survival for patients in CR was 27 months (range, 6-42), and those not in CR was 16 months (range, 6-53) (p=0.20). In addition, we observed that 75% of patients who attained CR are alive compared to 11.1% of those not in CR (p = 0.052). Conclusion: We demonstrate a superior CR rate of 30.7% with enasidenib amongstIDH2mutated RR-AML patients compared with clinical trial data showing CR rate of 19.6% (Stein et. al., 2019) despite our patients being heavily pre-treated with median number of two prior therapies (range; 1-5) including prior AHSCT in 2 patients. Moreover, a subset of patients (2 of 4 in CR) were able to undergo ASCHT. Finally, the achievement of CR at 2 months after initiation of therapy serves as an appropriate time point for response assessment. Disclosures No relevant conflicts of interest to declare.

Background: Oncolytic virotherapy is a novel immunomodulatory therapeutic approach for relapsed refractory hematologic malignancies. The Indiana strain of Vesicular Stomatitis Virus (VSV) has been engineered to encode interferon beta (IFNβ) and sodium iodine symporter (NIS) to produce VSV-IFNβ-NIS. The virally encoded IFNβ serves as an index of viral proliferation and enhances host anti-tumor immunity. The NIS is inserted as a reporter gene into the viral genome to enable noninvasive monitoring of viral spread using PET/CT imaging. Here we present results of Arm A (patients with low tumor burden) of the Phase I clinical trial (NCT03017820) of intravenous administration of VSV-IFNβ-NIS for patients (pts) with relapsed refractory hematological malignancies: MM, AML, and TCL. Methods: This was a classical 3+3 phase I trial, starting at 5x10^9 TCID50 (50% tissue culture infectious dose) level 1 through 1.7x10^11 TCID50 dose level 4 given as a single IV dose. The primary objective was determining the maximum tolerated dose of VSV-IFNβ-NIS alone; secondary objectives include estimating the safety profile and preliminary efficacy. Correlative objectives include monitoring the pharmacodynamics of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding and changes in the immune profile of peripheral blood leukocytes. Adverse events (AEs) are reported based on CTCAE v4; cytokine release syndrome (CRS) grading was based on Lee (Blood 2014; 124(2):188-195) criteria. Results: From April 2017, 12 pts have received IV VSV-IFNβ-NIS in Arm A: MM (7), TCL(4) and AML(1); 3 pts were treated at each dose level (DL) 1 to 4. At DL1, there were five grade 3 hematologic AEs (leukopenia [1], neutropenia [1], lymphopenia [3]) and no grade 3+ non-hematologic AEs. At DL2, three grade 3 hematologic AEs (anemia [2], lymphopenia [1]), four grade 4 hematologic AEs (neutropenia [1], leukopenia [1], lymphopenia [1] and thrombocytopenia [1]). There was 1 event of grade 2 CRS at DL2, two grade 3 non-hematologic AEs (nausea [1], dehydration [1]). At DL3, there was one grade 4 hematologic AE (neutropenia [1]), three grade 3 hematologic AEs (lymphopenia [2], leukopenia [1]), and one grade 3 non-hematologic AE (vasovagal reaction [1]). One grade 2 and grade 1 CRS by Lee criteria were observed. At DL4, there were three grade 3 hematologic AEs (lymphopenia [1], neutropenia [1], thrombocytopenia [1]), and three grade 3 non-hematologic AEs (AST increase [1], skin infection [1], soft tissue infection [1])and two grade 2 CRS. There were no dose limiting toxicities. There was one partial response (DL2) and one complete response (DL4); 3 pts had stable disease, 7 pts progressed. The MM pts receiving VSV-IFNβ-NIS at DL4 had stable disease. Six of the 7 MM pts demonstrated transient reduction in serum level of involved free light chain (LC) after VSV-IFNβ-NIS infusion, with median 20.4% reduction of the involved LC within 48 hrs. Viremia was detectable in all pts at the end of infusion, to varying levels at 30 mins, 1, 2, 4, 24, 48h or 72 hrs post infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-VSV antibodies were present by day 29. The IFN levels were detectable within 30 minutes of infusion and peaked between 4 and 48 hrs. The pts with a diagnosis of TCL mounted higher hIFNβ levels within 48 hrs. Remarkably, the TCL pt with CR (DL4) mounted a peak hIFNβ response of 18213.3pg/ml at 48 hrs post infusion, approximately 15-fold higher than any other pt enrolled in the study; and highest neutralizing antibody titer (1:40960). Conclusion: VSV-IFNβ-NIS can be safely administered by intravenous infusion in pts with hematological malignancies, up to a dose level of 1.7 x 1011 TCID50, without dose limiting toxicities and no CRS higher than grade 2. In this small phase 1 study, pts with T-cell lymphoma demonstrated the strongest signal of response, with one confirmed partial response at DL2 and one complete response at DL4. IFNβ is an early biomarker of tumor susceptibility; the magnitude of IFNβ elevation appears to correlate with treatment response. There were no confirmed responses in pts with MM, but the 20.4% transient reduction in LC suggests that combination strategies with VSV-IFNβ-NIS may result in deeper and sustained responses in MM. Future trials investigating combination strategies with immune modulatory drugs are being planned. Disclosures Peng: Imanis: Other: Equity Ownership. Witzig:Karyopharm Therapeutics: Research Funding; Immune Design: Research Funding; Spectrum: Consultancy; Incyte: Consultancy; Acerta: Research Funding; MorphSys: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding. Dispenzieri:Intellia: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Alnylam: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Gertz:Celgene: Consultancy; Annexon: Consultancy; Sanofi: Consultancy; Appellis: Consultancy; Amgen: Consultancy; Medscape: Consultancy, Speakers Bureau; Physicians Education Resource: Consultancy; Data Safety Monitoring board from Abbvie: Membership on an entity's Board of Directors or advisory committees; NCI SPORE MM: Research Funding; Johnson and Johnson: Speakers Bureau; Springer Publishing: Patents & Royalties; Amyloidosis Foundation: Research Funding; Advisory Board for Proclara: Membership on an entity's Board of Directors or advisory committees; Alnylam: Consultancy; Ionis/Akcea: Consultancy; Spectrum: Consultancy, Research Funding; Prothena: Consultancy; DAVA oncology: Speakers Bureau; i3Health: Consultancy; Advisory Board for Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; International Waldenstrom Foundation: Research Funding. Dingli:Rigel: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; Alexion: Consultancy; Apellis: Consultancy; Karyopharm Therapeutics: Research Funding; Millenium: Consultancy; Sanofi-Genzyme: Consultancy. Kapoor:Cellectar: Consultancy; Celgene: Honoraria; Janssen: Research Funding; Sanofi: Consultancy, Research Funding; Amgen: Research Funding; Takeda: Honoraria, Research Funding; GlaxoSmithKline: Research Funding. Lin:Janssen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Legend BioTech: Consultancy; Juno: Consultancy; Bluebird Bio: Consultancy, Research Funding; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Merck: Research Funding; Takeda: Research Funding; Gamida Cells: Consultancy; Sorrento: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vineti: Consultancy. Kumar:Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Cellectar: Other; Carsgen: Other, Research Funding; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Merck: Consultancy, Research Funding; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Sanofi: Research Funding; Dr. Reddy's Laboratories: Honoraria; Adaptive Biotechnologies: Consultancy; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Kite Pharma: Consultancy, Research Funding; Novartis: Research Funding; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; MedImmune: Research Funding; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; Karyopharm: Consultancy; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; BMS: Consultancy, Research Funding; Genecentrix: Consultancy; Tenebio: Other, Research Funding. Russell:Imanis: Other: Equity Ownership. OffLabel Disclosure: This describes of administration of Vesicular Stomatitis Virus Genetically Engineered to Express NIS and human interferon, in Patients with Relapsed or Refractory Hematologic malignacies

Introduction Clonal cytopenias of undetermined significance (CCUS) is defined by the presence of somatic driver mutations/copy number variations in hematopoietic cells in patients with low blood counts, in the absence of morphological evidence for myeloid neoplasms. Patients with CCUS are often symptomatic and can be transfusion dependent (TD), with high rates of progression to myeloid neoplasms, especially myelodysplastic syndromes (MDS). In addition, similar to MDS, there are reports of CCUS patients responding to hypomethylating agents. Due to the lack of a formal diagnosis/morphological dysplasia, CCUS patients are often denied therapies or clinical trial enrollment. We carried out this study to validate our hypothesis that CCUS shares similar clinical and survival characteristics with lower risk MDS (LR-MDS). Methods CCUS patients were prospectively identified from the clonal hematopoiesis clinic, whereas LR-MDS patients were retrospectively identified from our institutional database. LR-MDS was defined as very low, low and intermediate risk MDS based on the R-IPSS categorization. Baseline demographics, blood counts, bone marrow (BM) morphology, cytogenetics, and NGS results were abstracted. Transfusion dependency was defined as requiring at least one unit of red cell or platelets every 4 weeks. The Mann-Whitney-U and Fischer's exact test were used to compare quantitative and qualitative data in subgroups. Kaplan-Meier overall survival (OS) estimates were used for survival analysis and compared using the log-rank test. Results 186 patients were included in the study; 74 (40%) with CCUS and 112 (60%) with LR-MDS, median age 66 years, with 63% being male (Table 1). In the CCUS group, 93% had one or more mutations (67% >1 mutation) detected by NGS, while 7% had clonal cytogenetic abnormalities. Common mutations in CCUS included TET2 (30%), SRSF2 (20%), DNMT3A (13%) and ASXL1 (11%); with 28% of patients being red cell TD and 15% being platelet TD. On application of the R-IPSS stratification, 40%, 45%, 12% and 3% were in the very low, low, intermediate and high risk categories, respectively. LR-MDS subtypes included MDS-RS (50%), MDS-MLD (21%), MDS-EB (10%), MDS del5q (7%), and MDS-U (12%). SF3B1 mutations were seen in 67% (95% of MDS-RS), while TET2 and DNMT3A mutations were seen in 29% and 21%, respectively. 60% of LR-MDS patients had >1 mutation. 44% were red cell TD, while 10% were platelet TD. In comparison to patients with CCUS, LR-MDS patients were more likely to have higher white blood cell counts (p=0.002), higher neutrophil counts (p=0.009), higher platelet counts (p<0.001), be red cell TD (p=0.031), have BM ring sideroblasts (p<0.001), have higher BM blast% (p=0.004) and carry SF3B1 mutations (p<0.001); whereas CCUS patients were more likely to have higher monocyte counts (p=0.009) and more likely to carry IDH1 (p=0.005), ZRSR2 (p=0.005) and ATM (p=0.012) mutations. Importantly there were no differences with regards to R-IPSS cytogenetic risk groups, R-IPSS prognostic categories, platelet TD, and mutations involving TET2, DNMT3A, SRSF2 and ASXL1 and TP53. Notably signal pathway mutations and mutations involving tumor suppressor genes were infrequent (<5%) in both groups. The LR-MDS patients had a longer median follow up (53 vs 15 months) and at last follow up 72 (64%) and 11 (15%) deaths had been documented in the LR-MDS and CCUS groups respectively. 13 (18%) CCUS patients progressed to MDS (10) and AML (3) over a median of 15 months, while 9 (8%) LR-MDS patients progressed to higher grade MDS (1) and AML (8), respectively (median follow up 53 months). There was no difference in the median survival between CCUS (median OS not reached) versus LR-MDS (median OS 8.3 years) (p=0.372, Figure 1). Conclusion In spite of subtle phenotypic/molecular differences between LR-MDS and CCUS, both entities had similar prognostication, distribution of high risk mutations and survival outcomes. Within a short follow-up, 18% of CCUS patients progressed to MDS/AML, indicating that regardless of the absence of dysplasia these entities are a continuum of myeloid neoplasms. In order to improve access to treatments and clinical trial opportunities, we recommend that close consideration be given to consider CCUS as a MDS subtype. Disclosures Shah: Dren Bio: Consultancy.

Introduction: Truncating mutations in Additional Sex Combs-Like 1 (ASXL1) are associated with a high-risk disease phenotype in myeloid malignancies. In chronic myelomonocytic leukemia (CMML), truncating ASXL1 mutations are known to increase transcriptional activity of leukemic driver genes and have been associated with gene body hypermethylation. We interrogated the transcriptome and methylome of patients with ASXL1-mutant (MT) and -wildtype (WT) CMML using a multi-omics approach to test the hypothesis that gene expression is mediated through gene body methylation. Methods: Bone marrow mononuclear cells from patients with ASXL1 WT (n=8) and MT (n=8) CMML were subjected to targeted NGS of DNA, whole transcriptome shotgun sequencing (RNA-seq), and immunoprecipitation of DNA methyl residues (DIP-seq). After quality control all samples were sequenced on an Illumina HiSeq 4000 before further processing and data analysis. Differential gene expression analysis was performed to identify genes up-regulated in MT CMML. The samples in the two groups were treated as biological replicates and subjected to a consensus peak calling strategy requiring an overlap of at least 30% between samples and an adjusted p-value < 5x10-5 for a methylation peak to be considered statistically significant. For validation purposes methylation analysis was performed on 3 ASXL1 MT and 3 WT CMML patients using Illumina Infinium MethylationEPIC microarrays and differentially methylated regions were identified using a bump hunting strategy. Gene body methylation was defined as methylation in gene bodies (outside the promoter region, i.e. transcription start site ±2kb). Gene body methylation was compared between WT and MT CMML for the up-regulated genes and correlated with expression of all genes in MT CMML. Results: Sixteen WHO-defined CMML patients were included, median age 69 years (48-77), 63% male, 50% had truncating ASXL1 frame shift mutations. Abnormal karyotypes were observed in the same number of patients and the burden of co-mutations was similar between the two groups (median number per group 3 vs. 3, p=0.508). This included several modulators of DNA methylation including TET2, DNMT3A, and IDH2 (median number per group 1 vs. 1, p=0.699). There was a predominant up-regulation of gene expression in MT CMML: 707 genes up- and 124 down-regulated (FDR<0.050) without evidence of differential methylation in promoter regions of the differentially expressed genes. There was no difference in the number of genes with consensus methylation peaks in the gene body or the extent of gene body methylation (area under the consensus peaks) between MT and WT CMML for the differentially expressed genes (Figure 1a). We further validated these results using methylation microarrays (Figure 1b). Among the MT CMML patients, there was a curvilinear relationship between gene body methylation and gene expression across all genes with intermediate gene body methylation being associated with the highest gene expression (Figure 1c). As an alternative unbiased approach, we identified 1595 differentially methylated regions (DMR, regions with FDR<0.05) in the validation data set. We mapped all hypermethylated regions to gene bodies requiring that there was no concurrent hypermethylation of the promoter region of the same gene. With this approach we identified one of the 707 up-regulated genes (0.14%) with evidence of isolated gene body hypermethylation (HBZ, log2-fold change in gene expression 4.25, FDR=0.0008, DMR area=1.61, DMR FDR=0.018). Representative examples of up-regulated mitotic kinases without evidence of differential gene body methylation are shown in Figure 1d (pie charts represent the mean β-values of microarray probes, p-values represent the comparisons of mean β-values between MT and WT CMML per gene). Conclusions: Gene body methylation was positively associated with gene expression in MT CMML. However, the lack of differential gene body methylation between WT and MT CMML for the up-regulated genes make it an unlikely explanation for the observed increase in transcriptional activity among patients with MT CMML. Figure 1 Disclosures Ordog: Millipore Sigma: Patents & Royalties.

Background Clonal cytopenia of undetermined significance (CCUS) is a newly described entity with a high probability to progress into myeloid disorders upon follow-up (Malcovati et al, Blood 2017). Little data is known on how to effectively treat patients with CCUS. We hereby describe a single institution experience in managing symptomatic CCUS patients. Methods Patients who had CCUS and underwent active treatment were identified after receiving IRB approval. CCUS diagnosis was rendered if patients had non-diagnostic bone marrow biopsy evaluation combined with evidence of pathogenic myeloid somatic mutation using our institution next generation sequencing (NGS) panel (OncoHeme, Mayo Clinic). "Symptomatic patients" is determined by patients who need therapy beyond blood transfusion. Treatment type is based on physicians' choice. Clinical and lab results were collected. Response and progression were graded based on the MDS IWG 2006 criteria. The date of treatment initiation was used for overall survival (OS) and progression (either to myeloid neoplasm or worsening cytopenias) free survival (PFS) calculation. Stata 14.1(StatCorp) was used for data analysis. Results 1) Cohort characteristics: Between 2015 and 2020, 24 patients met inclusion criteria with median age of 72 years (range: 24-87). Twenty (83%) were men. 15 (63%) were white, 1 (4%) African American, 1 (4%) Native American and 7 unknown (33%). Eighty percent had ECOG PS≤1. Four (17%) patients had prior other hematology disorders and 3 (13%) had solid tumor. Two (8%) patients had previous radiation therapy, 2 (8%) received chemotherapy and 1 (4%) received allogeneic stem cell transplantation. At the time of treatment, the median of hemoglobin was 8.9 (range 6.8-13.7) g/dL, white blood cell 2.9 (range 1.4-12.3) *109/L, and platelet 76 (range 8-407) *109/L. Red blood cell (RBC) and platelet transfusion dependency rate were 54% and 29%, respectively. We identified 23 different mutations. Most common mutations occurred in SRSF2 (N=5, 21%), TET2 (N=5, 21%), U2AF1 (N=5, 21%), and TP53 (N=4, 17%). Ten (42%) patients had 1 mutation, and 14 (58%) had ≥2 mutations. Treatment included hypomethylating agents (HMA) (N=10, 42%), growth factors (N=11, 46%), steroid (N=4, 17%), testosterone (N=2, 8%), cyclosporine (N=2, 8%), rituximab (N=1, 4%), immunoglobulin (N=1, 4%), vitamin B12 and iron (N=1, 4%). Seven (29%) patients received ≥2 treatments. Median follow up duration was 17.5 months (range 8.9-26.1). Median time from the diagnosis to treatment was 2.1 months (range 0-26.8). 2) Molecular correlation with response The overall response rate (RR) was 50% (hematological or symptomatic improvement). Most responders (67%) were treated with HMA (p=0.013). HMA treatment effect was not significantly associated with DNA methylation genes (DNMT3A, IDH1, IDH2 and TET2, all p>0.05). The RR was 100% for the three patients with IDH1 mutation (treated with HMA, steroid and erythropoietin stimulating agent (ESA), respectively). The RR was 100% for the 3 patients with ZRSR2 mutation (2 HMA, 1 testosterone). The RR was 0% for 3 patients with SF3B1 mutation (1 testosterone and ESA, 1 vitamin B12 and iron, and 1 HMA). The number of mutations had no impact on RR (p=0.41). Table demonstrates the RR for each treatment. 3) Survival and progression outcome At the last day of follow up, RBC and platelet transfusion dependency rate were 38% and 25%, respectively. The median PFS was 16.9 months (95% CI: 7.5-65.3). Six patients (25%) progressed to myeloid malignancy with 4 myelodysplastic syndrome (MDS) and 2 acute myeloid leukemia (AML). Five patients progressed with worsening cytopenias. Among the 4 MDS patients, they had SF3B1, TET2, and ASXL1 mutations at the diagnosis of CCUS. Within the 2 AML pts, 1 had RUNX1 mutation, 1 had BCOR, DNMT3A, PHF6, RUNX1, SF3B1, STAG2, and TET2 mutations at the diagnosis of CCUS. The median OS was not reached with an estimated 2 year OS 77%. Three (13%) patients progressed with cytopenia and died. Their NGS at CCUS diagnosis showed ASXL1, TP53 and U2AF1 mutations, respectively. Conclusion Patients with CCUS responded to available treatments used for myeloid diseases. HMA were effective in this cohort. Mutation profile may have an impact on treatment response. Studies of larger cohorts are needed to validate our findings. Additionally, development of response and progression criteria would be helpful for such patients for reporting treatment outcomes. Disclosures Shah: Dren Bio: Consultancy.

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (selinexor), a first in class CRM1 inhibitor recently approved for multiple myeloma and diffuse large B-cell lymphoma (DLBCL), has been limited by substantial adverse effects (AEs). To address this clinical problem, we focused on identifying novel strategies to boost the potency, reduce toxicity, and broaden the applicability of CRM1 inhibitors to a wider range of malignancies. We discovered that salicylates could markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. KPT-330 was chosen as the prototypical CRM1 inhibitor given its current FDA approval status and characterized pharmacokinetics; and choline salicylate (CS) was chosen as the prototypical salicylate given its favorable pharmacokinetics and reduced antiplatelet, renal, neurological and gastrointestinal AEs in humans compared to other salicylates. By using cell lines belonging to different hematologic malignancies and solid tumors, we demonstrated ex vivo that the combination of KPT-330 and CS (K+CS) could induce unique and significant antitumor effect at much lower dose of KPT-330 (at 25% of the dose used in the clinic), thereby potentially mitigating prohibitive clinical AEs (Figure 1a-d, and e-g). This significant synergetic antitumor effect observed with K+CS ex vivo was also validated in vivo by using an NSG mouse model of mantle cell lymphoma (Figure 1h). Moreover, the K+CS combination did not show this potent toxic effect on non-malignant cells in vivo and was safe without inducing toxicity to normal organs in NSG mice. Mechanistically, protein profiling through mass spectroscopy revealed that K+CS uniquely affects the cellular pathways of DNA damage repair, DNA synthesis and cell cycle progression. Studies involving immunoblotting, cell cycle analysis, immunofluorescence microscopy assessing nucleocytoplasmic molecular export and DNA damage, reporter assay to assess homologous recombination repair proficiency and immunohistochemistry showed that, compared to KPT-330 treatment alone, K+CS decreased the expression of CRM1, Rad51 and thymidylate synthase proteins in vitro and in vivo, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, and importantly a unique cell cycle arrest in S-phase, thus leading to cell apoptosis. These effects on cellular proteins and pathways were unique to K+CS treatment and were not observed with KPT-330 or CS single agent treatment. Pathway analyses through RNA sequencing also paralleled the findings of proteomic studies thus further validating the unique effect of K+CS treatment on the aforementioned cellular pathways. Importantly, K+CS treatment exerted unique and significant antitumor effect ex vivo on primary malignant cells obtained from patient's with high-risk hematologic malignancies such as double hit DLBCL, BTK and BCL2 inhibitor resistant mantle cell lymphoma, TP53 deleted/mutated chronic lymphocytic leukemia and high-risk multiple myeloma thus signifying its broader applicability as a treatment option for these aggressive hematologic malignancies where there is a dire need to find new treatment strategies (Figure 1i). In summary, we describe a unique all-oral drug combination with a novel constellation of mechanisms of action on cellular pathways that are exploited by cancer cells. K+CS represents a new class of therapy for multiple cancer types and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for therapy of blood cancers. Disclosures Witzig: Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Incyte: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy; Immune Design: Research Funding; Spectrum: Consultancy.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Background: Chronic myelomonocytic leukemia (CMML) is a rare chronic myeloid malignancy with a poor prognosis. There are few epidemiologic studies on CMML. We studied population-based outcomes in CMML using the Surveillance, Epidemiology, and End Results (SEER) and the National Cancer Database (NCDB). Methods: The SEER 18 registries and NCDB Participant User File were used to identify patients with ICD-O-3 diagnosis code 9945/3 from 2004-2015. Incidence was identified using SEER and age-adjusted to the U.S. 2000 standard population. Causes of death were obtained and CMML-related death was defined as death from any myeloid disorder (CMML, acute monocytic leukemia, acute myeloid leukemia [AML], chronic myeloid leukemia, other myeloid/monocytic leukemia, and aleukemic, subleukemic, and NOS) to avoid misattribution. Relative survival (RS) was defined as the ratio of the proportion of observed survivors in a cohort of CMML patients to the proportion of expected survivors in a comparable set of individuals that did not have CMML, adjusting for the general survival of the US population for race, sex, age, and time when the diagnosis was established. Time to AML was calculated using the left-truncated life tables session and a 3 month latency period was used to prevent misattribution. Per the NCDB's system on classifying treatment, hydroxyurea, decitabine, and azacitidine are considered chemotherapy. The Kaplan-Meier method was used to calculate overall survival (OS), and Cox regression model was used to identify predictors of survival. Variables significant in univariate analysis (age, Charlson Deyo score [CDS], insurance type, and treatment facility type) were included in a multivariate analysis. Statistical analyses were performed using SAS version 9.0. Results: In the SEER database (n=4437), the median age at diagnosis was 76 years (interquartile range [IQR] 68-83) and 2,783 patients (63%) were male. Incidence rates (1 case/1,000,000 individuals) were as follows: overall 4.4, male 6.6, female 2.9, Non-Hispanic White 4.9, and Non-Hispanic Black 3.3. The incidence did not significantly change from 2004 to 2015. With a median follow up of 5.8 years (95% CI: 5.5-6.3), the median OS was 1.3 years (95% CI: 1.3-1.4). 3635 patients (82%) died. Among those who died, 2016 patients (55%) deaths were CMML-related. When comparing CMML patients to the US general population, 3,156 patients were matched to the expected survival tables. In the general U.S. population, the expected survival for 12, 24, 36, 48 and 60 months was 95.6%, 91.3%, 87.1%, 83.1%, and 79.2%, respectively. In contrast, the RS for patients with CMML at the same time points was 63.6%, 46.2%, 35.2%, 28.8%, and 23.1% (Figure 1). 229 patients (5.2%) progressed to AML. The median time to AML was 1.2 years (IQR 0.6-2.3). In NCDB (n=6403), the median age at diagnosis was 74 years (IQR 66-82). With the median follow up of 6 years (95% CI 3.5-9), the median OS was 1.3 years (95% CI 0.4-3.3). The distribution of CDS score 0, 1, and >1 was 4518 (71%), 1193 (18%), and 692 (11%) respectively. 4687 (77%) had government insurance, while 1399 (23%) had private insurance. 3750 (60%) were treated at a non-academic center. The year of diagnosis was associated with improved OS (HR 0.99, 95% CI: 0.98-0.99). Patients who were diagnosed in 2012-2015 had improved OS compared to patients diagnosed in 2004-2007 (HR 0.89, 95% CI 0.83-0.95) with OS at 1- and 5- years being 60% and 16% (2012-2015) vs. 56% and 17% (2008-2011) vs. 53% and 15% (2004-2007) respectively. 3029 (48%) patients received chemotherapy, while 270 (4%) patients received stem cell transplant as the first-line therapy. The median time to chemotherapy from diagnosis was 19 days (IQR 6-43). OS at 1-, 5-, and 10-years was 57%, 18%, and 4% (no chemotherapy), 56%, 14%, and 7% (received chemotherapy), 79%, 44%, and 38% (received stem cell transplant), and 56%, 15%, 5% (no stem cell transplant). OS curves are shown in Figure 2. Factors independently predicting inferior OS were age ≥ 65 years at the time of diagnosis (p<0.001), CDS ≥ 1 (p<0.001), government insurance (p<0.001), and treatment at a non-academic center (p<0.001) (Table). Conclusion: Despite an improvement in survival over the years, CMML continues to be a cause of significant morbidity and mortality. Older age, CDS ≥ 1, government insurance, and treatment at non-academic facilities were predictive of inferior survival. Disclosures Shah: Dren Bio: Consultancy.

Introduction: Additional Sex Combs-Like 1 (ASXL1) is a chromatin modifier frequently affected by truncating mutations in myeloid malignancies. In chronic myelomonocytic leukemia (CMML), truncating ASXL1 mutations are associated with the overexpression of leukemogenic driver genes, however the molecular mechanisms underlying this transcriptional activation remain controversial. We performed single-cell chromatin accessibility studies of ASXL1-mutant (MT) and -wildtype (WT) CMML to identify cis-regulatory elements (CREs) such as distal enhancer elements that may explain the observed transcriptional activity in MT CMML. Methods: Bone marrow mononuclear cells from a patient with WT and a patient with MT CMML were subjected to single-cell DNA transposase accessibility studies (scATAC-seq). The cryopreserved cells were thawed and resuspended and approximately 100000 viable mononuclear cells per sample were subjected to transposase assays before proceeding to single-cell partitioning into gel beads in emulsion, barcoding, library construction, and sequencing following an established 10X Genomics workflow. The target cell recovery was approximately 2000 cells per sample. Genomic libraries were sequenced on an Illumina HiSeq 4000 before demultiplexing, alignment to the reference genome, and post-alignment quality control. The 10X Genomics Cell Ranger ATAC software was used for demultiplexing, alignment of the reads to the GRCh38 reference genome, filtering and quality control, counting of barcodes and unique molecular identifiers, identification of transposase cut sites, detection of accessible chromatin peaks, count matrix generation for peaks and transcription factors, clustering, and differential accessibility analysis. The two-sample Kolmogorov-Smirnov test for equality of distribution functions was used to compare the distributions of single-cell entropies. Single-cell cis-co-accessibility scores were estimated using Cicero. Results: To mitigate potential effects on the analysis introduced by unequal cell recovery due to experimental conditions, we randomly sampled 1500 single cells from each genotype. Visualizing the first two principal components of the chromatin accessibility data revealed the differences in chromatin conformation between genotypes and also the increased heterogeneity within the MT sample (Figure 1a). To quantify this increase in intratumoral heterogeneity of chromatin accessibility in MT CMML, we calculated the single-cell entropies for both genotypes. There was an increase in the dispersion of single-cell entropy values (mean 0.793±0.026 versus 0.811±0.012, p=6.19x10-151) in MT compared to WT disease (Figure 1b). To leverage the power of the single-cell chromatin accessibility data, we estimated cis-co-accessibility scores (two-way chromatin interactions). There were 631 genes involved in two-way chromatin interactions that were either specific to MT CMML or were shared between MT and WT CMML and showed an increase in co-accessibility in MT CMML. Co-accessibility was higher in MT compared to WT CMML, regardless whether the chromatin interaction was MT-specific or shared between the genotypes (Figure 1c). Gene ontology analysis revealed increased chromatin accessibility in genes involved in the regulation of cell cycle, nuclear division, chromosome organization, cell cycle phase transition, DNA conformation change, and DNA replication (FDR<1.00x10-5). The construction of cis-co-accessibility networks demonstrated a remodeling of the chromatin landscape with MT-specific chromatin interactions involving key mitotic kinases such as MAP2K3 (Figure 1d). Conclusions: MT CMML is characterized by an increase in intratumoral heterogeneity and increased chromatin accessibility. The increased chromatin co-accessibility involved key mitotic kinases and may serve as a plausible explanation for the increased transcriptional activity observed in MT CMML. Figure 1 Disclosures Ordog: Millipore Sigma: Patents & Royalties.