ALBERT

Description: Background: Management of anemia is a common therapeutic challenge in patients with myelodysplastic syndromes (MDS). Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014). Aims: This is an ongoing, phase 2, multicenter, open-label study to evaluate the effects of luspatercept in patient (pts) with low-intermediate risk MDS. Endpoints included erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI, ≥ 8 weeks), duration of HI-E, pharmacodynamic and iron metabolism biomarkers, safety, and pt-reported QoL. Methods: Inclusion criteria included age ≥ 18 yr, Hgb 〈 10 g/dL (if 〈 4U RBC/8 weeks), no prior HMA, and no current lenalidomide or erythropoiesis-stimulating agent (ESA). An expansion cohort of up to 56 patients was added to this phase 2 study to evaluate response to luspatercept in pts who do not qualify for the phase 3 MEDALIST trial (for RS+ positive patients with baseline EPO ≥ 200 U/L and ≥ 2U RBC/8 weeks). These include pts with low transfusion burden (〈 4U RBC/8 weeks) who are either 1) ring sideroblast (RS)+ (≥ 15% RS in bone marrow) with baseline EPO ≤ 200 U/L and no prior ESA use, or 2) RS- with any baseline EPO level and any prior ESA use. Patients are treated with 1.0 mg/kg of luspatercept every 3 weeks for up to 5 doses, with titration up to 1.75 mg/kg. Patients may rollover to an open-label extension study for up to an additional 2 years of treatment. Results: Results for the initial patient cohorts have demonstrated a high proportion of HI-E and RBC-TI responses in RS+ patients. Data for the additional ESA-naïve RS+ patients with low EPO levels and RS- patients with 3 months of treatment will be presented at the meeting. Conclusions: Erythroid response to luspatercept has been demonstrated in RS+ patients with lower-risk MDS and is being explored in ESA-naïve RS+ patients with low EPO levels and RS- patients. A Phase 3 study of luspatercept in regularly-transfused RS+ patients with lower-risk MDS according to IPSS-R is ongoing (MEDALIST study; NCT02631070). Disclosures Donovan: Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership. Giagounidis:Celgene Corporation: Consultancy.

Description: Introduction Mantle cell lymphoma (MCL) is a rare but often aggressive subtype of non-Hodgkin's lymphoma, with many patients experiencing short survival. While both first and subsequent-line therapies are available, it is unclear what health economic evidence is available for treatments. To provide context for economic evaluations, it is also important to understand the economic burden of MCL and the quality of life which patients experience. Our study aims to comprehensively understand health economic (costs and resource-use, and economic evaluations) and health-related quality of life (HRQoL) evidence base for patients with MCL through three systematic literature reviews (SLRs). Methods Search strategies were designed to capture studies reporting economic or HRQoL outcomes for patients with MCL. Searches were date-limited in order to identify studies published in the last 10 years (2007-current). The following electronic databases were searched: MEDLINE, Embase, NHS Economic Evaluation Database (NHS EED), and EconLit. In addition, we searched congress abstracts (at ASCO, ASH, ISPOR, AMCP, EHA, and ESMO proceedings) presented over the previous 2 years. Potential publications were screened in duplicate by 2 reviewers. Additional online searches were carried out to identify health technology assessment (HTA) submission documents reporting health economic evaluations for patients treated with pharmacological interventions. All searches were conducted in October 2017, and were updated in March 2018. Results The SLR identified 11 economic evaluations reported across 16 publications, and 7 studies reporting data for costs or resource-use. Table 1 presents a summary of the economic evaluations that reported incremental cost effectiveness ratios (ICERs). Four of the 11 economic evaluations presented models for patients in the first-line setting, and 7 presented models for patients in the relapsed/refractory (R/R) setting.. The majority of economic evaluations were conducted using a Markov model with 3-5 health states. R-Chemo was the most common comparator used in both the first line and relapsed/refractory settings. Across the 7 studies reporting costs or resource-use data, all 7 reported data for resource-use and 3 additionally reported costs data. There were a variety of costs and resource use data reported, including treatment-specific and non-treatment-specific data. Studies reported adverse events (AEs) as key drivers of increased costs and resource use. One study specifically reported statistically significant (p≤0.005) increases in emergency room visits were associated with MCL disease related AEs (odds ratio [OR]: 10.571). The following disease-specific HRQoL measures were reported: FACT-Lym, FACT-G, and EORTC QLQ-C30. There was little consistency in the measures used to evaluate HRQoL across studies. Table 2 presents the 2 studies reporting FACT-Lym scores. In both studies, trends for improvement in FACT-Lym total scores following treatment were reported. One phase 3 randomized study in R/R MCL reported 66% of ibrutinib-treated patients and 48% of temsirolimus-treated patients achieving a clinically meaningful improvement in FACT-Lym score. A second study in front line MCL reported improvements in the FACT-Lym total score following treatment with lenalidomide + rituximab. Each of these studies also demonstrated that clinical response to treatment was associated with the improvement in overall HRQoL. Conclusions These SLRs highlight the limited availability of published economic and HRQoL evidence for patients with MCL. The paucity of evidence is even more evident when first-line and R/R data are examined separately. Markov models with 3-5 health states represented the majority of economic evaluations . In both the first line and relapsed/refractory settings, R-Chemo was the most common comparator used. For both settings, AEs may have significant effects on the economic burden MCL places on healthcare payers. Novel agents have shown a clinically meaningful improvement in HRQoL. The noted economic burden of the disease demonstrates a need for newer treatments that decrease the burden MCL places on health care systems globally. There remains a need for future research to further understand the economic and HRQoL burden of MCL. Disclosures Monga: Janssen Pharmaceutica NV: Employment. Garside:Janssen Pharmaceutica NV: Employment. Davids:Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy; Surface Oncology: Research Funding. Tam:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ward:Janssen Pharmaceutica NV: Consultancy. Fotheringham:Janssen Pharmaceutica NV: Consultancy. O'Donovan:Janssen Pharmaceutica NV: Consultancy. Parisi:Janssen: Employment. Tapprich:Janssen Pharmaceutica NV: Employment.

Description: Bruton's Tyrosine Kinase (BTK), a TEC-family non-receptor tyrosine kinase, plays a critical role in B-cell development and function. Targeting of BTK with covalent inhibitors like ibrutinib has become a standard approach for many B-cell malignancies, including chronic lymphocytic leukemia (CLL), Waldenstrom macroglobulinemia, mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). Yet, many patients demonstrate intrinsic or acquired resistance to covalent BTK inhibition. The prognosis of patients who relapse after ibrutinib treatment for MCL or CLL is dismal, highlighting the urgent need for new approaches that overcome resistance to current BTK inhibitors. We hypothesized that degradation of BTK could be a better alternative to inhibition alone, as it would both: 1) maintain efficacy in cells harboring the ibrutinib-resistant BTK C418S mutation and 2) target non-catalytic functions of BTK. To address this, we turned to a small molecule-mediated protein degradation platform that utilizes an E3 ligase-targeting moiety linked to the ligand of a target of interest, so that the target can be marked for ubiquitination and subsequent proteasomal degradation. We previously showed that BTK is one of the most robustly degraded kinase targets using a nonspecific kinase inhibitor linked to a imide-based core followed by agnostic proteomics. We synthesized highly potent and selective degraders of BTK using imides as a base. To do so, the parent BTK inhibitor CGI1746 was linked to thalidomide using either polyethylene glycol (DD-03-007) linkers or saturated hydrocarbon chain (DD-03-171) linkers. After verifying that these degraders induce dimerization of BTK and CRBN and penetrate cells, we explored the pharmacological effects in vitro. Both DD-03-007 and DD-03-171 reduced BTK levels at concentrations as low as 40 nM within 4h of treatment. Furthermore, cellular BTK levels remained low for 24h after washout, showing that these degraders are capable of sustaining depletion of BTK for an extended period of time (Figure). DD-03-007 and DD-03-171 are both ligase and proteasome-dependent, as shown by co-treatment with bortezomib or MLN-4924 as well as competition experiments with lenalidomide or CGI-1746. Moreover, the degraders exhibited strong synergy with the HCK inhibitor A419259, suggesting that it would be possible to recapitulate ibrutinib's previously reported polypharmacology with an HCK inhibitor. We overexpressed wild-type or C481S BTK in TMD-8 cells and ran an antiproliferation assay, which showed that DD-03-007 overcame ibrutinib resistance associated with BTK C481S mutation. Next, we explored the effects of the degraders in MCL specifically. Proteomic analysis showed that DD-03-171 is a triple-degrader, as it degrades BTK but retains degradation activity on IKZF1 and IKZF3. Finally, we performed In vivo efficacy studies in cell line and patient-derived xenograft (PDX) models of MCL. The latter was obtained from a patient who had progressed on ibrutinib. In both models, DD-03-171 caused a significant reduction in tumor burden at an early timepoint. DD-03-171 also markedly extended survival compared to treatment with ibrutinib or lenalidomide alone (Figure). In conclusion, we developed highly potent and selective BTK degraders with activity in vivo against human MCL that induce the degradation of multiple factors essential for MCL survival. Figure. Figure. Disclosures Treon: Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Johnson & Johnson: Consultancy; BMS: Research Funding. Weinstock:Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership.

Description: The covalent Bruton tyrosine kinase (BTK) inhibitor ibrutinib is highly efficacious against multiple B-cell malignancies. However, it is not selective for BTK, and multiple mechanisms of resistance, including the C481S-BTK mutation, can compromise its efficacy. We hypothesized that small-molecule–induced BTK degradation may overcome some of the limitations of traditional enzymatic inhibitors. Here, we demonstrate that BTK degradation results in potent suppression of signaling and proliferation in cancer cells and that BTK degraders efficiently degrade C481S-BTK. Moreover, we discovered DD-03-171, an optimized lead compound that exhibits enhanced antiproliferative effects on mantle cell lymphoma (MCL) cells in vitro by degrading BTK, IKFZ1, and IKFZ3 as well as efficacy against patient-derived xenografts in vivo. Thus, “triple degradation” may be an effective therapeutic approach for treating MCL and overcoming ibrutinib resistance, thereby addressing a major unmet need in the treatment of MCL and other B-cell lymphomas.

Description: Background: Management of anemia is a common therapeutic challenge in patients with MDS. Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014). Aims: This is an ongoing, phase 2, multicenter, open-label, long-term extension study to evaluate the effects of luspatercept in patients (pts) with low-intermediate risk MDS. Endpoints include long-term safety and tolerability, erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI, ≥ 8 weeks), duration of HI-E, pharmacodynamic and iron metabolism biomarkers, and pt-reported QoL. Methods: Inclusion criteria included age ≥ 18 yr, Hgb 〈 10 g/dL (if 〈 4U RBC/8 weeks), ESA refractory or EPO 〉 500 U/L, no prior HMA, and no current lenalidomide or ESA. Luspatercept was administered SC every 3 wks for up to 5 doses in the base study (NCT01749514), including 7 dose escalation cohorts (n=27 total, 0.125 to 1.75 mg/kg) and an expansion cohort (n=31, starting dose 1.0 mg/kg, max 1.75 mg/kg). A 2-year extension study (n=32) is ongoing (NCT02268383). Results: Data (as of 4 Mar 2016) were available for the 32 extension study pts. Of these, 13 pts received 〈 4U RBC/8 weeks pretreatment (low transfusion burden, LTB) and 19 pts received ≥ 4U RBC/8 weeks (high transfusion burden, HTB). Median age was 72 yr (range 29-90 yr), 59% had prior ESA. Median Hgb for LTB pts was 8.5 g/dL (range 6.4-10.1 g/dL) and median RBC transfusion burden for HTB pts was 6 U/8 weeks (range 4-14 units). 91% pts were RS+ (≥ 15% RS in bone marrow). IWG HI-E was achieved in 11/13 (85%) LTB pts and 15/19 (79%) HTB pts. 11/22 (50%) pts with at least 2 units transfused in 8 weeks prior to dosing with luspatercept achieved RBC transfusion independence for at least 8 weeks. The range of transfusion independence was 9 to 80+ weeks, with most responders still receiving treatment. IWG HI-E response rates were 83% for RS+ pts, 90% for EPO 〈 200 U/L, 86% for EPO 200-500 U/L, and 50% for EPO 〉 500 U/L; 85% for ESA-naïve and 79% for those who had prior ESA treatment. RBC transfusion independence was achieved in 58% for EPO 〈 200 U/L, 50% for EPO 200-500 U/L, and 33% for EPO 〉 500 U/L. Luspatercept was well tolerated, with 3 related grade 3 adverse events of myalgia, worsening of general condition, and blast cell count increase. The most common related AEs (≥ 2 pts in both base and extension studies) were fatigue, bone pain, diarrhea, myalgia, headache, hypertension, and injection site erythema. Conclusions: Long-term treatment with luspatercept was well tolerated and led to erythroid response in 81% of low-intermediate risk MDS pts who enrolled into the extension study. A Phase 3 study of luspatercept in regularly-transfused RS+ patients with lower-risk MDS according to IPSS-R is ongoing (MEDALIST study; NCT02631070). Disclosures Platzbecker: Onconova, Teva, Celgene, Janssen, Novartis, Amgen: Honoraria, Research Funding. Donovan:Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership. Giagounidis:Celgene Corporation: Consultancy.

Description: Introduction: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by erythroid hypoplasia. It is associated with a number of congenital anomalies and a high risk of developing specific cancers. DBA is caused by germline mutations or deletions in genes affecting ribosomal biogenesis and function, with autosomal dominant or X-linked recessive patterns of inheritance. The most commonly mutated gene is RPS19, seen in approximately 25% of patients. About 45% of DBA families have no known disease-causing pathogenic variant. Methods: Affected and unaffected individuals from families with DBA were ascertained through the IRB-approved NCI IBMFS retrospective/prospective cohort study (ClinicalTrials.gov Identifier: NCT00027274). Study participants completed detailed family and medical history questionnaires, medical records were reviewed, and a subset of families underwent clinical evaluations at the NIH Clinical Center. DBA patients enrolled prior to 2014 underwent routine clinical mutation testing for the established DBA genes; beginning in 2014, DBA patient samples (buccal and blood DNA) were evaluated by whole exome sequencing (WES) for mutation identification. We incorporated WES with deletion analyses and copy number variant (CNV) assessment to uncover the genetic changes causative of DBA. Deletion analyses performed included SNP genotyping and array comparative genomic hybridization. Functional effects of the genetic variants were proven by pre-rRNA processing defect analysis by Northern blot. Controls for functional studies were healthy mutation-negative individuals from the IBMFS study. Results: Genetic testing information was available in 61 of the 87 families with DBA enrolled in the IBMFS study. Thirty-five of the 61 families did not have a known genetic cause at enrollment. Our combined approach of WES, deletion and CNV analyses identified the causative pathogenic variant in 18 of the 35 (51%) uncharacterized DBA families. We discovered pathogenic variants in two previously undescribed genes in two DBA families. One family had a nonsynonymous variant (p.K77N) in RPL35; the second family had a nonsynonymous variant (p. L51S) in RPL18. Both of these variants result in characteristic pre-rRNA processing defects. Our analyses also uncovered germline mosaic deletions in known DBA genes in both buccal and blood cells of two patients from two different families. One was a 1.8 Mb mosaic deletion in chromosome 15 including RPS17; the other was a large 2.5 Mb mosaic deletion on chromosome 3 including RPL35A. In addition to these findings, we found variants in previously known DBA-associated ribosomal genes in 14 of the 35 families. We further evaluated the genomic characteristics of the entire DBA cohort. Pathogenic variants in ribosomal DBA genes were found in a total of 44 of the 61 families (72%) on whom genetic testing information and/or biospecimens were available. RPS19 was the most frequently mutated gene and accounted for 36% of families, followed by RPL35A and RPS26, accounting for 14% and 11% each, respectively. Notably, 30% of the variation in disease-causing genes in our cohort was due to a single copy or mosaic gene deletion. We had complete parental testing and inheritance information on 23 (52%) of the 44 families whose gene was identified. Ten of the 23 (43%) had an inherited mutation and 13 (57%) had a de novo change in the causative gene (both parents were negative for the affected child's disease-associated mutation). At this time, 17 of 61 families tested (28%) do not have a characterized disease-associated mutation. Conclusion: This efficient comprehensive genomic approach was the basis for our discovery of two novel causes of DBA, characterization of ribosomal gene deletions not previously described to be disease-associated, and of DBA-associated germline mosaicism. We identified the disease-associated mutations in 51% (18 of 35) of our families without a known genetic cause of DBA. A total of 74% (44 of 61) of our families are now genetically characterized. Our comprehensive approach appears to provide more genomic information than other methods since pathogenic variants of DBA genes have been reported previously in about 55% of DBA patients. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p

Description: Background: Selective BCL2 inhibition with venetoclax, induces deep responses in relapsed MM. However, acquired resistance to venetoclax frequently occurs. Importantly, in the Bellini trial, while the combination of venetoclax and bortezomib doubled the median progression free survival compared to control, it resulted in shorter overall survival. These results highlight the challenges secondary resistance poses and the need to investigate the mechanisms mediating the emerged resistance to venetoclax. Methods & Results: Bone marrow (BM) aspirates were collected from patients (n=8) treated with venetoclax prior to initiation of therapy and at disease progression. Mononuclear cell fractions were isolated through ficoll coupled with magnetic sorting of CD138+cells. Ex-vivo functional profiling of venetoclax sensitivity was performed on CD138+cells. Unbiased mRNA and DNA profiling was conducted by single-cell RNA-sequencing (scRNA-seq), single cell ATAC-seq and single cell copy number analysis (scCNV) using the GemCode system (10x Genomics). Cell Ranger, Seurat, Signac and Monocle were used for data analysis. Ex-vivo apoptosis studies revealed a dynamic shift of the IC50s of venetoclax from a median of 100 nM in pretreatment sample to 〉1000 nM at disease progression. Single cell CNV profiling identified a significant expansion of a pre-treatment subclonal (70%) at the time of relapse. The scale of the 1q CNV gain varied from 3 Mb to a very focal gain of 100 kb encompassing the MCL1 gene locus. Of note copy number gain was also identified in the BCL2L1 gene, but no copy number losses were detected in the BAX or BAK gene loci. Single cell RNA profiling of the same samples confirmed the gain in the MCL1 mRNA transcript with downregulation of BCL2 at the time of relapse. Consistent with a branching evolutionary model, scATAC-seq revealed increased chromatin accessability studies at the MCL1 locus in subclones of relapsed samples. Cell trajectory analysis and pseudotime ordering of cells (Monocle) revealed the emergence of a highly proliferative clone and of an MCL1 dependent clone as the disease evolved from its original BCL2 dependent cluster at pseudotime (t0) (Figure). Furthermore, ex-vivo apoptosis profiling revealed a shift in the sensitivity of the CD138+cells with an acquired sensitivity to MCL1 inhibitor (S63845). These findings suggest that the 1q21 copy number gain with MCL1 upregulation is likely mediating the acquired venetoclax resistance. In order to functionally confirm whether the gain in MCL1 is sufficient to shift BCL2 to MCL1 dependency and induce resistant to venetoclax, we stably overexpressed MCL1 in the KMS12PE BCL2-dependent MM cell line (KMS12PE_MCL1) and examined its sensitivity to venetoclax and/or S63845 relative to control vector (KMS12PE_EV). Of interest, co-immunoprecipitation (co-IP) of BIM in KMS12PE_MCL1 demonstrated a shift in BIM loading and co-IP with MCL1 and BCL2 while it was restricted to BCL2 in KMS12PE_EV cells. Importantly, venetoclax IC50 of KMS12PE_EV increased by 200 folds in KMS12PE_MCL1 cells with acquired sensitivity to S63845. scCNV analysis of one patient did not identify any gain in the 1q locus at the time of disease progression to venetoclax. Mutation analysis however identified a de novo BCL2 mutation [NM_000633.2:c.332A〉C, p.(Asp111Ala)]. To determine whether the Asp111Ala mutation alone is sufficient to confer resistance to venetoclax, the mutant was overexpressed in KMS12PE cells. While BIM binding to BCL2 was unaffected, Aps111Ala largely abrogated venetoclax-induced BIM displacement from BCL2 and reduced KMS12PE cells sensitivity to venetoclax by ~7.5 folds. Structure modeling also demonstrated the inability of venotoclax to tightly bind mutant BCL2_Asp111Ala hydrophobic groove. Conclusion: We have discovered and functionally characterized a novel BCL2 mutation that confers in vitro and in vivo resistance to venetoclax-treated MM patients. In addition we have identified through single cell profiling an enrichment of MM clones with MCL1 locus copy number gain at the time of acquired venetoclax resistance. Early detection and dynamic monitoring of these abnormalities (BCL2 mutant or 1q gain) with early therapeutic interventions targeting these clones may enhance venetoclax efficacy and improve patients' survival. Figure Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Bahlis:Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Description: Background: Targeting the anti-apoptotic BCL2 protein in haematological malignancies has demonstrated significant anti-tumoral activity in a subset of multiple myeloma patients harbouring rearrangements involving the CCND1 and the immunoglobulin heavy chain enhancers (Eμ and α1/2). The mechanisms underlying the dependency of this subgroup of MM patients on BCL2 remains to be elucidated as well as the mechanisms of resistance to BCL2 inhibition with BH3 mimetic venetoclax. Methods and Results: Sorted bone marrow plasma cells from a cohort of t(11;14) myeloma patients treated with venetoclax were profiled through multi-omics single cell mRNA expression (scRNAseq), copy number profiling (scCNVseq) as well as chromatin accessibility with single cell ATAC-seq. Sequenced reads were aligned to hg38 reference genome. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Monocle, Signac, and Cicero R packages. Single plasma cells exhibited differential chromatin accessibility landscapes within and across individual patients as well as pre- and post-venetoclax with enrichment of MYC:MAX, RELA, IRF family, RUNX1/3 and ETS motifs. Integration of mRNA and ATAC data revealed a dynamic change of regulatory motifs across individual cell clusters with evidence of selective pressures driven by venetoclax treatment. Similarly mRNA profiling of the apoptotic genes pre- and post-venetoclax exposure showed loss of BCL2 and upregulation of MCL1 and/or BCL2L1 as well as loss of the BH3-only pro-apoptotic genes PMAIP1 and BCC3 in single cell clusters. mRNA levels mirrored open chromatin at the gene bodies and their respective promoter loci consistent with a direct transcriptional regulation. In a patient with several fold upregulation of the BCL2L1 transcript in the post-venetoclax sample (Figure A-B), scATACseq identified a gain in the chromatin accessibility mapping to a genomic region centromeric to BCL2L1 locus on chromosome 20 (chr20:31,617,200-31,619,900). Single cell CNV analysis identified a 5q loss (chr5:142,400,001-156,240,000) mapping to NR3C1 locus explaining with the clinical resistance to dexamethasone. Importantly scCNV also revealed a copy number gain mapping to the same locus with the newly acquired chromatin accessibility on chromosome 20. Mate-pair analysis of the sequencing reads identified the potent IGLL5 B-cell enhancer on chromosome 22 (chr22:22,960,001-22,980,000) as the mate partner juxtaposed the BCL2L1 locus (Figure C). This finding explains the robust upregulation of BCL2L1 mRNA observed in this patient and the shift in BCL2 dependency detected by ex vivo BH3 sensitivity profiling. Of note, while scCNV analysis also depicted a gain in 1q21 (chr1:149,940,001-169,980,001) MCL1 locus at the time of venetoclax resistance the acquisition of t(20,22) shifted the plasma cells dependency to BCL-xL rather than MCL1. This finding was corroborated by the plasma cells ex vivo resistance to dual BCL2 and MCL1 inhibition. Conclusion: Dynamic single cell epigenome and transcriptome profiling of pre- and post-venetoclax of primary plasma cells identified a de novo translocation driving BCL-xL transcription with the IGLL5 B-cell enhancer. This demonstrates that in addition to canonical TF-promoter regulation, restructuring of immunoglobulin regulatory sequences (i.e., enhancers) can also drive aberrant malignant circuitry endowing resistance to anti-BCL2 agents. Figure. Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Bahlis:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Description: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome, characterized by congenital anomalies, bone marrow failure and cancer predisposition. FA patients with mutations in FANCB exhibit X-linked inheritance pattern. FANCB patients have been frequently associated with a severe phenotype, resembling VACTERL association with hydrocephalus, although heterogeneity in these patients still exists and is not well understood. To determine the underlying pathogenesis of clinical heterogeneity, we investigated phenotype-genotype correlations in 19 patients from 16 families with FANCB pathogenic variants. We also investigated functional properties of five FANCB missense variants to determine their possible contribution to clinical manifestations. Patients were accrued at the Rockefeller University Hospital in the International Fanconi Anemia Registry. Among the 16 families with FANCB pathogenic variants, three carry nonsense variants, five have indels, two with large deletions, one has an intragenic duplication, two with canonical splice site variants that should result in aberrant splicing, and five display missense variants. One patient carried both an indel and a missense variant. Patients with variants leading to FANCB truncations (including nonsense variants, indels, large deletions/duplications, and splice variants) tend to show shorter survival and earlier hematologic onset, than patients with missense variants. However, the differences did not reach statistical significance due to small sample size. Functional properties of five missense variants were examined by overexpression of variant cDNA (HA-tagged) in FANCB-null fibroblasts, followed by assessment of FANCD2 ubiquitination, mitomycin C (MMC) sensitivity, FANCD2 foci formation and HA-FANCB localization. We found that expression of p.W479G and p.L676P FANCB variants leads to near-normal FANCD2 ubiquitination upon MMC exposure, and MMC sensitivity. Conversely, p.L43S-expressing cells behaved like FANCB-null fibroblasts. Expression of p.L329P and p.G750V resulted in low levels of FANCD2 ubiquitination upon MMC exposure and intermediate MMC sensitivity. Expression of p.L43S resulted in smaller-sized and lowest quantity of FANCD2 foci with predominant cytoplasmic localization of HA-FANCB, while other variants showed intermediate phenotype. We quantified the nuclear expression of HA-FANCB, which confirmed the lowest relative nuclear expression of p.L43S variant, suggesting a defect in nuclear localization necessary for the interstrand crosslink repair. We also assessed splicing defects in the patient primary cells by reverse transcription PCR. Majority of the transcripts in the patient carrying c.1435T〉G (predicted to express p.W479G) variant showed aberrant splicing, exon 7 skipping, which should result in an out-of-frame truncated protein. Although the expression level of c.1435T〉G encoding the missense variant p.W479G is lower than the splicing variant, its functional characteristics were near-normal, thus reflecting relatively milder phenotype in this patient. Overall patient survival correlated with residual function of their FANCB variants as assessed by in vitro assays described above. In summary, we report clinical course and mutation types of 19 patients with FANCB pathogenic variants. We also report that FANCB missense variants can have varying degree of residual function, which correlates with patient survival. Our data suggest that variant-level analysis may be a useful prognostic marker of patient outcome. In vitro assays evaluating the residual function of proteins with missense variants may help to predict patient outcome. Disclosures No relevant conflicts of interest to declare.