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Background: The inherited bleeding disorder (IBD) community has witnessed significant advances in care, yet important gaps persist, particularly in rare disorders and underserved populations. An initiative spearheaded by the National Hemophilia Foundation (NHF) and shaped by the patient community aims to accelerate progress through a national research blueprint. The blueprint is being designed to identify and guide research priorities toward those areas that most significantly impact the lives of individuals affected today and articulate clearly defined opportunities to make the greatest impact for the future. Methods: NHF has enlisted individuals with IBDs as subject matter experts (SMEs) to guide this initiative by elevating the most pressing issues affecting them today and informing expert discussions on actionable research priorities for the future. SME insights have been collected through listening sessions, a cross-community survey, and participation in multi-disciplinary working groups. The NHF State of the Science Research Summit (SOS) in September 2021 will address the input of the working groups and will also feature patient vignettes to illustrate today's unmet needs and contextualize the research priorities identified to address them. As part of this interactive Summit, SMEs from traditionally underrepresented patient populations are also being enlisted to participate in remote participation groups (RPGs) with the goal of soliciting input that further tailors the research priorities to the needs of these populations. The RPGs will be comprised of individuals with bleeding disorders or their caregivers who represent specific populations by race/ethnicity (for example, African Americans, Asian Americans, Indigenous persons, Mexican and Central American Hispanic individuals, etc.). Within each group, NHF will aim to include individuals with diverse experiences based on their IBD, barriers related to access to care, gender and sexual orientation. During each session, the moderated RPGs will participate in the live summit, discuss the expert dialogue, and share real-time perspectives and comments about how the content addresses, or not, their specific community needs. The expert SOS panel will then have the chance to address their comments. The commentary from these sessions will be included in the NHF blueprint to define the research path forward for the community. The RPGs are also expected to be reconvened in Spring 2022 to review and improve upon the opportunities identified in the blueprint. Results: NHF has enlisted broad and diverse community support to ensure the blueprint accurately represents the opportunities to create meaningful and lasting impact for individuals with IBDs. In total, 42 patients and caregivers participated in listening sessions; 125 contributed to the community survey; 15 are participating in the Summit working groups and approximately 200 are being enlisted for the remote participation groups. The themes to be addressed during the SOS reflect the input provided by the SMEs and health professionals (see Table 1). Conclusions: Actively soliciting the patient community's views is central in our process to advance research in IBDs. By enlisting the participation of historically underserved community segments, this effort aims to address some of the most persistent and pressing issues affecting the IBD community today. Specific insights from the RPG participation in the Summit will be included in the presentation. This blueprint, which will guide the U.S. research community, could help fundamentally redefine the experience of diverse populations living with these disorders. Figure 1 Figure 1. Disclosures Witkop: Teralmmune, Inc.: Consultancy. Recht: Octapharma: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy; Takeda: Consultancy; uniQure: Consultancy; Foundation for Women and Girls with Blood Disorders, Partners in Bleeding Disorders: Speakers Bureau; American Thrombosis and Hemostasis Network: Current Employment; Oregon Health & Science University: Current Employment; Kedrion: Consultancy; Hema Biologics: Consultancy; Genentech: Consultancy; CSL Behring: Consultancy; Catalyst Biosciences: Consultancy. Valentino: Spark: Ended employment in the past 24 months.

Unrepaired DNA double-strand breaks (DSBs) pose a serious threat to genomic stability, potentially leading to the formation of oncogenic mutations, as well as translocations, deletions and amplifications. Almost all multiple myeloma (MM) patients are characterized by such copy number alterations and structural variations. Therefore, we evaluated and report that MM cells have increased ongoing spontaneous DNA damage which could be exploited to create synthetic lethality in combination with inhibitors of homologous recombination (HR). We evaluated various types of DNA damage in peripheral blood mononuclear cells (PBMCs)from 15healthy controls, 4 MM cell lines and CD138+ bone marrow plasma cells (BMPCs) from 16 patients with monoclonal gammopathy of undetermined significance, 18 with smoldering myeloma (SMM) and 15 with MM. Relative to normal PBMCs, MGUS and MM cells showed significantly higher levels of endogenous DNA breaks (measured as olive tail moments with single-cell gel electrophoresis), abasic sites (Elisa-based detection) as well as DNA break repair efficiency (all P 70% inhibition of HR activity measured using plasmid-based assay. Inhibition of HR by CYT-0178 was also demonstrated in U2OS cell using DRGFP (Addgene) assay. Moreover, treatment of RPMI-8226, MM1S, H929 MM cell lines with CYT-0851caused a dose-dependent inhibition of HR activity as assessed by homologous strand exchange assay. To evaluate the impact of these drugs on cell viability, MM cell lines (U266, RPMI8226 and MM1S) and normal cells were incubated with CYT-0851 and CYT-0178 at various concentrations and durations. Treatment with CYT-0851 induced dose- and time-dependent cytotoxicity in all myeloma cell lines tested with minimal impact on normal cells. For CYT-0178, the treatment at 10 𝛍M for seven days resulted in 50% to ~80% cell death in MM cell lines tested, although lower (5 𝛍M) dose had no significant impact. We further evaluated combination of these inhibitors with conventional MM drugs melphalan, cyclophosphamide and lenalidomide,for impact on cytotoxicity. Treatment with CYT-0851 led to a synergistic anticancer effect when combined with these chemotherapeutic agents in both the MM cell lines tested. Increased cytotoxicity of lenalidomide and melphalan was also observed by CYT-0178 in MM1S cells. In summary, we demonstrate that MM cells have ongoing endogenous DNA damage which provides basis for synthetic lethality in combination with HR inhibitors while sparing normal cells. Moreover, novel RAD51 inhibitors CYT-0178 and CYT-0851 inhibit HR activity, impair cell growth and increase efficacy of existing drugs in multiple myeloma. Disclosures Day: Cyteir Therapeutics: Employment. Munshi:Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy.

Activation Induced Cytidine Deaminase (AICDA/AID) is a DNA-directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. In cancer cells, AID causes significant genotoxic stress through DNA replication fork damage, creating a dependency upon the homologous recombination repair factor, RAD51, for survival. We have demonstrated anti-cancer activity through disruption of this axis in multiple preclinical lymphoid cancer models. Autoreactive B cells depend on RAD51 for survival and are chronically auto-stimulated and therefore continually re-express AID. It has been shown that ectopic expression of AID in autoreactive B-cells causes genome-wide DNA damage (similar to cancers). Given the role of autoreactive B cells and autoantibodies in autoimmune disorders, we hypothesize that immunomodulation of B cells via the RAD51/AID axis will remediate inflammatory disease processes. Our previous data suggests that RAD51 modulation enhances the CD73+ B cell population and reduces antibody diversity in T1D mice, indicating precise effects on AID-mediated antibody diversification. CYT-0853 is a novel RAD51 inhibitor that sensitizes cells to AID activity. Here, we assessed the in vivo effect of CYT-0853 on primary B cells and antibody production. Wild-type C57BL/6 mice were treated with 40mg/kg CYT-0853 or vehicle for five weeks. One-week post-treatment start, mice were immunized with DNP-KLH antigen mixed with Complete Freund's Adjuvant. A second booster with DNP-KLH antigen mixed with Incomplete Freund's Adjuvant was administered two weeks later. At termination, blood, spleen, and bone marrow was collected for analysis by flow cytometry. Surface expression of CD45, CD19, IgM, and IgG1 was assessed to determine white blood cell count, B cells, and pre- and post-class switch recombination (CSR), respectively. While no significant changes to B cell populations were observed in bone marrow or spleen, we demonstrate that CYT-0853 significantly decreases the median number of circulating CD45+ and IgG1 (post-CSR) B cells (61.8% vs. 31.6% and 8.7% vs. 4.4%, respectively). In addition, we observed a modest, significant increase in the amount of IgM+ (pre-CSR) B cells. These results were complemented by an associated overall significant decrease in circulating IgM levels. Of note, no adverse effects were observed in these mice over this treatment period. Based on these data and the role of B cells not only in antibody production, but also as antigen-presenting cells in multiple sclerosis, we tested our molecule in the myelin oligodendrocyte glycoprotein35-55-experimental autoimmune encephalomyelitis model of multiple sclerosis. Prophylactic treatment using 40mg/kg CYT-0853 did not affect disease activity or circulating cytokine production, however we observed a significant decrease in the spleen. Based on these results, further exploration is warranted to harness the power of CYT-0853 on the AID/RAD51 axis. This specific targeting may elicit beneficial therapeutic changes to B-lymphocyte populations and provide a novel immunomodulatory target to treat immunity and inflammation. Taken together, these data provide a foundation for continued preclinical development of CYT-0853 with applicability towards autoimmune diseases. Disclosures Aprahamian: Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership.

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. The homologous recombination (HR) repair pathway is an attractive target. HR deficient cancers are hypersensitive to numerous anticancer drugs, and tumors will often induce expression of HR genes to promote drug resistance. RAD51 is a key component of the HR pathway. RAD51 forms nucleoprotein filaments at sites of DNA damage and replication fork stalls, mediating homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We utilized four small molecule inhibitors of RAD51-mediated HR for evaluation of RAD51 as a potential therapeutic target. Compounds CYT-0851, CYT-0853, CYT-1027, and CYT-1127 were evaluated for anti-cancer activity in vitro and in vivo. To determine the impact of the small molecules on RAD51 and HR, all four were tested for effects on RAD51 focus formation and sister chromatid exchange (SCE) activity. All the compounds showed a reduction in SCE activity, however only CYT-0851 and CYT-0853 produced a measurable reduction in RAD51 foci. We have previously shown that that RAD51 inhibition leads to accumulation of DNA breaks, and ultimately cell death, in cells expressing the DNA mutator protein Activation Induced Cytidine deaminase (AICDA/AID). Cytotoxicity assays were performed in an AID+ (Daudi, Burkitt's Lymphoma) and AID- (WI-38, fibroblast) cell lines. All four compounds were preferentially active in AID+ cells with little to no cytotoxicity observed in the AID-negative WI-38 cell line. CYT-0853 was the most potent in the Daudi cell line with an EC50 of 8nM. All four compounds were orally bioavailable in all preclinical species tested but showed differences in pharmacokinetics. Preclinical cell line derived xenograft models of AID-high Burkitt's lymphoma (Daudi) and B-cell acute lymphoblastic leukemia (CCRF-SB) were used to determine the in vivo anti-tumor activity of the compounds in lymphoid cancer models. CYT-0851 and CYT-0853 both showed significant anti-tumor activity with tumor growth inhibition of greater than 50% in both models. Further analysis showed drug exposure with CYT-0851 was more consistent in the CDX models than CYT-0853. Overall, these data indicate that RAD51 and HR are attractive therapeutic targets for the treatment of lymphoid malignancies and that CYT-0851 is a viable clinical development candidate. Disclosures Day: Cyteir Therapeutics: Employment. Maclay:Cyteir Therapeutics: Employment. Cyr:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership.

We have developed CYT01B, a novel RAD51 inhibitor, that sensitizes cells to Activation Induced Cytidine Deaminase (AID) activity. In cancer cells, AID causes significant genotoxic stress through DNA replication fork damage, creating a dependency upon the homologous recombination repair factor, RAD51, for survival. CYT01B acts by destabilizing RAD51 focus formation, leading to its premature nuclear export and degradation. We have shown CYT01B to be effective in AID expressing cells, however, we had yet to address if inhibition of RAD51 could act as a sensitizer to current therapeutics. To determine potential drug combinations, a matrix study was performed with CYT01B (concentration range of 20nM to 5μM) and six different targeted agents or chemotherapeutics in three different cell lines: ARPE19/HPV16 (HPV immortalized normal epithelial cell line), KYSE-70 (head and neck cancer cell line) and Daudi (Burkitt's Lymphoma cell line). We then used the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). The compounds tested were the ATR inhibitor VE-822 (concentration range of 39nM to 2.5μM), the RPA inhibitor TDRL-505 (concentration range of 39nM to 5μM), the proteasome inhibitor Bortezomib (concentration range of 39nM to 2.5μM), Carboplatin (concentration range of 156nM to 10μM), and the PARP inhibitors Olaparib and Niraparib (concentration range of 78nM to 5μM). With VE-822 we observed synergy in the KYSE-70 cell line with ambiguous results in Daudi and ARPE19/HPV16. In ARPE19/HPV16 cell line, synergy was observed with CYT01B at 39nM with all concentrations of VE-822, but antagonistic activity was seen at the high and low concentrations. In Daudi, antagonism was observed at the highest concentrations of VE-822, but an additive effect was noted at the lower concentrations of VE-822. Antagonism was observed at all concentrations of CYT01B with TDRL-505 in both Daudi and ARPE19/HPV16. Weak synergy was observed in KYSE70 cells at 156 and 312nM CYT01B. CYT01B was synergistic with Bortezomib in ARPE19/HPV16 at all concentrations but was consistently antagonistic in KYSE-70 and Daudi. We observed synergy with carboplatin in all cell lines, with the effect consistent across the full concentration range in the cancer cell lines. Synergy was also observed consistently across the full concentration range in all three cell lines with both PARP inhibitors. However, Olaparib showed a stronger synergistic effect than Niraparib. These data suggest that CYT01B may be effective as a combinatorial therapy with platinum based chemotherapeutics and with PARP and ATR inhibitors. Overall, we conclude that there is significant potential for RAD51 inhibition to be used in future combination cancer treatment strategies and warrants further exploration in vivo. Disclosures Maclay: Cyteir Therapeutics: Employment. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction: Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from mutations in the gene encoding α-galactosidase A (GLA). It is characterised by the abnormal accumulation of neutral glycosphingolipids (GSL), predominantly Globotriaosylceramide (Gb3), in the lysosomes of multiple cell types including vascular endothelial cells. It is associated with early-onset stroke, cardiomyopathy, and progression to end-stage renal failure. Current standard of care for FD is enzyme replacement therapy but alternatives including allogeneic stem cell transplantation have also been explored. Fabry disease is a good model for liver-targeted gene therapy, as relatively low levels of enzyme correction may suffice to reduce storage. Additionally, a broad therapeutic window exists, and mouse models of FD enabling preclinical proof-of-concept studies are readily available. Here we report the preclinical results of adeno-associated virus (AAV) mediated, liver-targeted gene transfer of a self-complementary (sc) vector encoding a codon-optimised GLA transgene under transcriptional control of a hepatocyte-specific promoter pseudotyped with AAV8 capsid (scAAV2/8-LP1-GLAco). Methods: In this study a single dose of scAAV2/8-LP1-GLAco vectors were administered into the tail vein of homozygote GLA knockout Fabry mice at either 1 month or 3 months of age. Two doses were tested for each age group; 2x1012 vg/kg and 2x1013 vg/kg, and an additional age-matched group left as untreated to serve as an experimental control for the effects of treatment. Mice were followed in the treatment for up to 11 months before sacrificing at 11 or 14 months of age. To assess the kinetics and durability of transgene expression, plasma GLA levels were measured at various time intervals post injection. Results: Long-term follow-up of Fabry mice injected with scAAV2/8-LP1-GLAco vector demonstrated sustained, durable levels of plasma GLA throughout the typical lifespan of a mouse. Plasma GLA activity levels at 10 or 11-months post vector administration to 1 and 3-month-old mice respectively, demonstrated a dose response from 2 x 1012 to 2 x 1013 vg/kg of >1 log increase in expression. These GLA activity levels represent supra-physiological expression (x 4000 of normal levels) which resulted in uptake of GLA from the bloodstream into kidney and cardiac tissues leading to dose-dependent clearance of lipid deposits in these tissues as analysed by electron microscopy. Most importantly, sustained expression of GLA resulted in complete normalisation of Gb3 and lyso-Gb3 levels in plasma and liver tissues as quantified by mass spectrometry. Interestingly, these preclinical studies in Fabry mice at the age of 1 and 3 months demonstrated not only prevention but also reversal of lysosomal storage deposits through liver-specific expression of GLA. Finally, in these in vivo mouse studies no adverse clinical signs were observed, despite the increased plasma exposure to GLA. Conclusions: Collectively, these data provide strong evidence that our liver-directed AAV-mediated gene therapy approach holds considerable therapeutic potential for the treatment of Fabry disease. We anticipate that a single dose IV procedure will pose minimal burden to Fabry patients and will be a viable alternative to biweekly enzyme infusions, potentially reducing treatment-related morbidity whislt improving patient quality of life and potentially providing them with a functional long-term cure. Disclosures Kia: Freeline: Employment, Equity Ownership. McIntosh:Freeline: Consultancy. Hosseini:Freeline: Employment, Equity Ownership. Sheridan:Freeline: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties; Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The B-cell specific DNA mutase/recombinase Activation Induced Cytidine Deaminase (AID) initiates DNA double strand breaks (DSB) necessary for immunoglobulin class switch recombination in activated B-cells. However, AID-induced DSBs are not restricted to immunoglobulin loci, and we previously demonstrated that the homologous recombination (HR) factor RAD51 is critical to repair widespread AID-initiated DSBs. While it is known that failure to properly repair AID-induced genomic DSBs is cytotoxic to B-cells, the pathways culminating in cell death were not fully known. Here, we demonstrate that AID-initiated DSBs can trigger mitotic catastrophe, a non-apoptotic cell death mechanism. Using genetic and chemical biology approaches we further show that AID-induced mitotic catastrophe involves multiple pathways, including mTOR. We also demonstrate that AID-induced cytotoxicity can be used to effectively and preferentially target AID-expressing lymphoid malignancies, in xenograft cancer model. This study reveals a cell death response to genomic damage by AID, providing new insight into the mechanisms that ensure cellular survival during B-cell maturation. Our findings support the idea that RAD51 modulation may be leveraged as a novel "synthetic lethal" therapeutic approach for treating AID-expressing malignancies, via the induction of mitotic catastrophe. Disclosures Mills: Cyteir Therapeutics, inc: Employment, Equity Ownership, Patents & Royalties. Hasham:The Jackson Laboratory: Patents & Royalties.

Introduction Recent developments of novel immunotherapeutic drugs have shown promising results for patients with hematologic malignancies, however, an unmet need for accurate and specific biomarkers persists. To address this need, we developed a novel integrative analysis procedure for the automated analysis of multidimensional flow cytometry data obtained from the peripheral blood of patients with chronic lymphocytic leukemia (CLL). State of the art flow cytometry analysis is accomplished by manual sequential segmentation, or gating, of cell populations based on similarities in fluorescence and light scatter characteristics through visualization of the data in one- or two-dimensional plots. This approach has a number of limitations, including the subjective nature of the gating and the inability to fully utilize the high-dimensional data. Recent efforts have produced sophisticated computational methods that overcome many of these limitations; however, these newer computational methods have not been rigorously tested in a clinical context and have focused on the rigorous and automated analysis of samples from individual patients, with substantially less effort towards the analysis of patient populations. The ultimate goal of our analysis is to develop computational approaches that will enable an identification of subsets of patients with distinct immunological markers. Methods We developed a novel analysis framework that facilitates automated identification of both common cell types and patient population subgroups, based on post-processing of individual sample analysis with the FLOCK program. FLOCK identifies clusters of putatively similar cells in an individual sample by multidimensional clustering of the fluorescence marker and light-scattering measurements. We developed a rigorous hierarchical clustering approach to identify common “cell signatures” across multiple patients. The cell signatures were then mapped back onto the individual patient samples and used in a second clustering that identified patient subgroups based on similar abundances of specific cell types. Results We used our analytic framework to analyze multidimensional flow cytometry data (26 cell surface markers in 4 different antibody cocktails) from peripheral blood specimens of a heterogeneous group of 55 CLL patients and 13 healthy controls. Our analysis revealed distinct differences between controls and CLL patients. Analyzing the non-malignant peripheral blood cell types, we were furthermore able to differentiate between distinct clinical subpopulations of patients (e.g. identify treatment-naïve patients from those that had previously undergone chemotherapy). Conclusion/Discussion Using a novel integrative analysis procedure to analyze complex flow cytometry data of the peripheral blood from CLL patients, we are able to identify distinct cell type distributions. We propose that this information is a marker for the overall health/disease status of the corresponding patient, and could ultimately be used for diagnosis, prognosis, and selection of optimal treatment. In the context of multiple novel treatment options for CLL patients, such a tool will be crucial for defining individual patient prognosis, and defining an accurately matched treatment plan. Disclosures: No relevant conflicts of interest to declare.

The BCR-ABL oncogene induces human Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) that advances to acute phase of CML called blast crisis. In this acute phase, CML patients can develop either B-ALL or acute myeloid leukemia. In B-ALL, differentiation of leukemic cells are blocked at pro-/pre-B stage, and the underlying mechanism is unknown. We hypothesize that this blockade of B-cell differentiation may be important for the development of B-ALL induced by BCR-ABL, and if so, promotion of B-leukemic cell differentiation would create a novel therapeutic strategy for B-ALL. To test this hypothesis, we first compared the percentages of IgM+ B-leukemic cells in BALB/c and C57BL/6 (B6) mice with BCR-ABL-induced B-ALL, because we have previously found that B-ALL develops more quickly in BALB/c mice than in B6 mice (Li et al, J. Exp. Med.189:1399–1412, 1999). We expressed BCR-ABL in bone marrow (BM) using retroviral transduction and transplantation in these two different strains of inbred mice to induce B-ALL. There were significantly more peripheral blood B220+ B cells in BALB/c B-ALL mice than those in B6 mice, correlating to faster B-ALL in BALB/c mice than in B6 mice. Among these B220+ cells, IgM+ cells were much less in BALB/c mice than in B6 mice. We also compared rearrangement of the B cell antigen receptor (BCR) heavy chains (m chains) between BALB/c and B6 backgrounds using BCR-ABL-expressing pro-B cell lines isolated from the B-ALL mice. Normal m chains rearrangement was found in B6 leukemic cells, but not in BALB/c leukemic cells. These results indicate that more differentiated B-leukemic cells are associated with less aggressive disease. To further demonstrate the role of blockade of B-cell differentiation in B-ALL development, we induced B-leukemic cell differentiation by co-expression of BCR-ABL and intact immunoregulatory tyrosine activation motifs (ITAM) contained in immunoglobulin (Ig)_/Igß complexes in BM cells of B-ALL mice, comparing to expression of BCR-ABL alone. We treated these mice with imatinib (orally, 100 mg/kg, twice a day). The treated mice with B-ALL induced by co-expression of BCR-ABL and ITAM lived three-week longer than those with B-ALL induced by BCR-ABL only, with some mice in long-term remission. Prolonged survival was associated with 50% increased B220+/IgM+ B-leukemic cells in peripheral blood of the mice. Taken together, our results demonstrate that blockade of B-cell differentiation is critical for the development of B-ALL induced by BCR-ABL, and provide a rationale for combination therapy of B-ALL with imatinib and induction of leukemic cell differentiation.