ALBERT

Description: SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Description: Multiple myeloma (MM) is a plasma cell disease that represents the second most common adult hematologic malignancy in the United States. Advances in the treatment of MM, including the development of proteasome inhibitors have improved clinical outcomes. However, many patients fail to respond to these newer agents or relapse after initial response. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression making it an ideal therapeutic target. Recent studies suggest that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors such as BCL-2 and displays significant activity against MM. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BRD4 antagonist JQ1 decreased the expression of c-MYC, BCL-2, and BCL-XL, diminished cell viability, disrupted clonogenic survival, and triggered apoptosis in a panel of MM cell lines. Subsequent assays conducted in MM cell lines confirmed a strong reduction in MYC levels and activity following treatment with JQ1 along with consistent increases in the expression of genes regulated by BRD4 including CDKN1A and HEXIM1. Interestingly, gene expression profiling assays revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with JQ1. Based on its ability to alter cellular survival capacity, we hypothesized that HDAC6 induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat) and evaluated the impact of each on the anti-MM effects of JQ1. Abrogation of HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with apoptosis induction as evidenced by enhanced caspase-3 cleavage and further reductions in c-MYC expression. These data suggest that HDAC6 induction may represent a key mechanism that promotes drug resistance and/or limits the efficacy of bromodomain inhibitor therapy. Our collective findings indicate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel and promising approach to augment the efficacy of bromodomain inhibitors for MM that warrants further investigation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Patients with relapsed or refractory (R/R) MYC-altered DLBCL have poor outcomes, and other than for a subset of patients who may benefit from chimeric antigen receptor T cell therapy, no treatment has shown a significant durable benefit or impact on survival outcomes. Fimepinostat is a first-in-class, well-tolerated, oral small molecule inhibitor of HDAC and PI3K enzymes. There is particular interest in evaluating fimepinostat in patients with MYC-dependent tumors as nonclinical studies demonstrated that fimepinostat inhibits transcription of MYC and a subset of MYC-associated genes. Additionally, MYC protein levels were downregulated by fimepinostat in part through inhibition of PI3K-mediated ubiquitination. Pharmacodynamic inhibition of HDAC and PI3K has also been demonstrated in human studies. Here we report the outcome of R/R DBLCL patients treated with fimepinostat in a Phase 1 and Phase 2 study, with an emphasis on outcomes for patients with MYC-altered disease. Patients and Methods A total of 105 R/R DLBCL patients were enrolled on the Phase 1 study CUDC-907-101 (n = 37) and the Phase 2 study CUDC-907-201 (n = 68). In CUDC-907-101, 14 patients were found to have MYC-altered disease, defined as presence of MYC rearrangement by either central or local testing by fluorescent in situ hybridization or MYC protein expression ≥40%) by immunohistochemistry (IHC). In CUDC-907-201, 46 patients had confirmed MYC-altered disease by central IHC testing. Across both studies, patients without available tissue or prior test results were deemed as MYC-status unknown (n = 23). Results A total of 19 responses (9 CR, 10 partial responses [PR]) were reported across both studies. The objective response rate (ORR) in MYC altered patients was 23.3% (14/60). Responses showed encouraging durability with a median duration of response of 13.6 months (range: 2.8 to Not Calculable [NC]). Five MYC-altered responses were ongoing as of the data-cut. Two MYC-altered patients achieving CR discontinued treatment early to pursue stem cell transplantation. Responses associated with fimepinostat often require multiple cycles of treatment to manifest (median time to first response = 2.5 months for MYC-altered patients), and of patients who were treated for ≥2 months, a large proportion (17/33; 52%) achieved a response. Patients with low disease burden at screening (tumor lesions diameters ≤ 5 cm and lactate dehydrogenase [LDH] 〈 1.5 x upper limit of normal [ULN]) generally continued treatment longer and were most likely to derive clinical benefit (Table 2). Conclusions The biologic rationale, tolerable safety profile, and evidence of durable anti-tumor activity in MYC-altered R/R DLBCL support the continued development of fimepinostat in this poor-prognosis patient population. Patients with low disease burden features may be more likely to have sufficient duration of drug exposure to allow clinical benefit. Future enrollment will focus on patients with screening characteristics most likely to derive the greatest benefit from fimepinostat treatment. Disclosures Landsburg: Takeda: Consultancy; Curis: Consultancy, Research Funding. Ramchandren:Seattle Genetics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Merck: Research Funding; Pharmacyclics LLC an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Lugtenburg:Millennium/Takeda: Consultancy, Research Funding; Servier: Consultancy, Research Funding; Roche: Consultancy; BMS: Consultancy; Sandoz: Consultancy; Genmab: Consultancy. Younes:Takeda: Honoraria; Abbvie: Honoraria; BMS: Honoraria, Research Funding; Novartis: Research Funding; Curis: Research Funding; Incyte: Honoraria; Seattle Genetics: Honoraria; Janssen: Honoraria, Research Funding; Bayer: Honoraria; Sanofi: Honoraria; Astra Zeneca: Research Funding; Celgene: Honoraria; J&J: Research Funding; Pharmacyclics: Research Funding; Roche: Honoraria, Research Funding; Merck: Honoraria; Genentech: Research Funding. Tuck:Curis, Inc: Employment, Equity Ownership. Barta:Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.

Description: Lenalidomide (LEN) has established a new paradigm of targeted therapy in MDS. The mechanistic underpinnings of LEN efficacy are related to the synthetic lethality of this agent through its ability to bind cereblon (CRBN). LEN induces degradation of CK1α, which is encoded by the CSNK1A1 gene located on the del(5q) CDR, whereby haploinsufficient levels of this gene allow for selective toxicity to deletion mutants. Another common cytogenetic abnormality present in patients with myeloid neoplasia (MN) is -7/del(7q). To date no selective therapies exist for -7/del(7q), an urgent unfulfilled need, given the poor prognosis associated with this cytogenetic abnormality. We were interested to explore if this same notion of selective toxicity may be possible in del(7q) myeloid patients and sought to screen drugs for this focused population. From a large cohort of patients with MN (n=3,328), we found -7/del(7q) in 10% (n=316) of patients. We first identified a signature pattern of haploinsufficient genes on -7/del(7q) based on NGS. We then searched for haploinsufficient genes which, if targeted by investigational drugs, could provide a therapeutic window for selected MN subtypes in analogy to LEN in del(5q). For the purpose of our analysis, haploinsufficient expression was defined as

Description: Abstract 1529 Introduction: The importance of bone marrow aspiration and biopsy in the evaluation of hematopoietic and non-hematopoietic disorders is well established. However, this technique is associated with morbidity and mortality risks.1 Recently, a battery-powered bone marrow biopsy system was developed to allow operators to safely, quickly and efficiently access the marrow space. We previously evaluated this device in swine models and in patients needing routine hematology outpatient evaluation.2 In the current study we compared the powered device to the traditional manual technique by relatively assessing pain scores, procedure times, biopsy capture rates, quality of material retrieved, safety and operator satisfaction. Methods: Two large academic medical centers participated in this trial (San Antonio, TX and Madrid, Spain). The study protocol was approved by each center's institutional review board. Adult patients requiring bone marrow biopsies were considered for the study. Following informed consent, patients were randomized to have procedures using a manual biopsy device (T-handle Jamshidi bone marrow biopsy and aspiration set, Cardinal Health, Dublin, OH) or the Powered device (OnControl 11 gauge/102mm Bone Marrow Biopsy System, Vidacare Corporation, Shavano Park, TX). After infiltration of the skin and medullary bone with local anesthesia, a visual analog scale (VAS) pain score was recorded immediately following skin puncture and once again at the end of the procedure for each patient. Procedure time was measured from skin puncture to core specimen ejection from the needle. Pathologic assessment of 30 randomized samples was carried out. Operator satisfaction with devices was measured on a scale of 0–10, with 10 as the highest rating. Statistics were calculated using t-test and chi-square, with an alpha-level of 0.05. Results: Five operators from 2 sites enrolled 50 patients (Powered, n=25; Manual, n=25). Of those patients, 58% were male and 42% were female; and had a mean age of 56.0±18.0 years. The mean height was 167.5 ± 10.5cm and the mean weight was 78.7 ± 22.7kg. Forty percent were lymphoma patients—the largest diagnostic group. Between patient groups, there were no significant differences in the means for these variables. See Table below for quantitative results, including pathology analysis. For the pathology qualitative analysis, there was no difference between groups for hemorrhage, clot/particle spicules, or smear spicules. Conclusions: Results of this trial suggest that the use of a Powered bone marrow biopsy device significantly reduces needle insertion pain. While not reflected in the results, overall pain may be better tolerated due to the important difference in procedure time. Moreover, the superior size and overall quality of core specimens retrieved by the Powered device provides more material for pathologic evaluation, thereby increasing diagnostic yield and reducing the need for repeat procedures. Cohesiveness of the medullary bone sampled was comparable for both techniques; however, the Powered system was less likely to recover non-hematopoietic tissue (e.g. cortical bone and soft tissue). Artifact was slightly more common with the Powered device (aspiration, hemorrhage and crush) but this did not impact on the diagnostic quality of the sample. No differences in safety data were noted for either technique and operator satisfaction favored the Powered device. 1. Bain BJ. Bone marrow biopsy morbidity and mortality. British Journal of Haematology 2003;121:949-51. 2. Swords RT, Kelly KR, Cohen SC et al. Rotary powered device for bone marrow aspiration and biopsy yields excellent specimens quickly and efficiently. J Clin Pathol 2010;63:562-5. Disclosures: Swords: Vidacare Corporation: Research Funding. Anguita:Vidacare Corporation: Research Funding. Kelly:Vidacare Corporation: Research Funding. Philbeck:Vidacare Corporation: Employment. Miller:Vidacare Corporation: Employment, Equity Ownership. Brenner:Vidacare Corporation: Research Funding.

Description: Autophagy contributes to therapeutic resistance and malignant progression by generating alternative metabolic fuel to maintain cell survival under stress conditions including those imposed by hypoxia, radiation, chemotherapy, and targeted agents. The FDA approved anti-malarial drug hydroxychloroquine (HCQ) inhibits autophagy through the disruption of lysosomal function. Robust efforts to repurpose HCQ for cancer therapy based on these properties stimulated numerous clinical trials where HCQ was combined with an array of other anticancer regimens and ultimately produced modest clinical activity. However, it was unclear if the maximum tolerated dose of HCQ was sufficient to completely inhibit autophagy in tumors. New autophagy inhibitors with increased potency and more favorable therapeutic indices are clearly needed to rigorously investigate the potential benefit of this approach. Polyvalent molecules can yield nonlinear, multifold potency against their respective targets compared to their corresponding monomers. We generated a series of novel dimeric compounds containing modified core elements of HCQ, CQ and the anti-schistosomal drug lucanthone with the goal of developing new autophagy inhibitors with superior potency, tolerability, and anticancer efficacy. Initial screens that tested for drug-induced increased expression of p62, a protein that is specifically turned over by autophagy, and anticancer activity identified ROC-325 as a lead agent. The structure of ROC-325 was confirmed by NMR and MS analyses. Direct comparison of the in vitro activity of ROC-325 and HCQ in 13 different genetically and histologically diverse human cancer cell lines demonstrated that ROC-325 was approximately 10-fold more potent than HCQ based on IC50 analyses. Acute myeloid leukemia (AML) was selected as a primary malignancy for intensive investigation based on the high sensitivity of FLT3-ITD+ MV4-11 cells to this agent in preliminary studies. Transmission electron microscopy analyses demonstrated that treatment with ROC-325 triggered all of the hallmark features of autophagy inhibition including the accumulation of autophagosomes with undegraded cargo, an increase in lysosomal membrane permeability, deacidification of lysosomes, and elevated LC3B, p62, and cathepsin D expression. Bafilomycin A1 clamp experiments showed that ROC-325 potently inhibited autophagic flux. Genetic impairment of two different genes that are essential for functional autophagy, ATG5 and ATG7, using lentiviral shRNA approaches significantly diminished the anticancer effects of ROC-325, thus indicating that autophagy inhibition is a key component of its anticancer mechanism of action.RNA sequencing and gene level analyses demonstrated that treatment with ROC-325 (1 µM) in MV4-11 cells altered the levels of autophagy-dependent degradation pathways while preserving protein synthesis through the upregulation of post-translational ribosomal, methylation, and splicing components. In vitro treatment of a panel of human AML cell lines and normal human bone marrow progenitors demonstrated that ROC-325 diminished AML cell viability (IC50 range 0.7-2.2 µM), antagonized clonogenic survival, and induced apoptosis in a manner that was therapeutically selective. Analysis of primary blasts from patients with AML showed that its activity was not significantly affected by adverse cytogenetics or multi-drug resistance due to relapsed/refractory clinical status. Oral administration of 50 mg/kg ROC-325 (QDx5) to mice bearing disseminated MV4-11 human AML xenografts significantly increased lifespan (P

Description: Background: We have previously reported on ethnic disparities in outcomes of multiple myeloma (MM) pts (pts) and shown that Hispanics have the shortest median overall survival (OS) and myeloma-specific survival (MMS) while Asians have the longest. Ethnic minorities are increasing in number in the United States (US) and have been historically underrepresented in population databases with limited follow up since the introduction of novel agents for MM. We did an updated analysis with longer follow up for ethnic minorities to explore the changes in outcomes by race and also explored MM incidence rates, not reported previously. Methods: Surveillance Epidemiology and End Results (SEER) 13 Registry data (1973-2013) for adult pts (〉18 yr) with confirmed diagnosis of MM was utilized. To avoid bias of under representation of ethnic minorities, analysis was restricted to pts diagnosed in 1992 or later. Years of diagnosis were 1992-1995 (pre-stem cell transplant; SCT), 1996-2002 (after SCT but before novel therapeutics) and 2003-2013 (after introduction of novel therapeutics). Cases that received a diagnosis at death certificate/autopsy, without follow-up records, lacking documentation on age at diagnosis, sex, or race/ethnicity were excluded. Cox proportional hazards models were used to evaluate association between patient characteristics and survival. All statistical tests utilized the SAS software (v9.4) and were two-sided with a significance level of 0.05. Results: The final analysis included 68431 MM pts (37300 males; 55%, 31131 females; 45%). Age-group cohorts included: 18-44 yr (2519; 4%), 45-54 yr (8131; 12%), 55-64 yr (15416; 23%), 65-74 yr (20214; 30%), and ≥75 yr (22151; 32%). Mutually exclusive racial subgroups included: non-Hispanic White (NHW; 44618, 65%), non-Hispanic African-American (AA; 13164, 19%), non-Hispanic Asian/Pacific Islanders (API; 3570, 5%), Hispanic (H; 6759, 10%) and Native American (NA; 320, 0.5%). Trends in age-specific incidence rates were similar for all races but except the 20-24 yr group, were highest for AA and lowest for API. (Figure 1) Survival analysis showed that females had a better median OS (3.4 vs 3 yr; p