Campos, Cecília Bonolo de

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNA) and mutations that are central to disease pathogenesis, prognosis, risk-stratification, and identification of response or resistance to therapies. Fluorescence in situ hybridization (FISH) is gold standard in the clinical laboratory for detecting prognostic CNAs in CLL (e.g. deletion 17p13 (del(17p), deletion 11q23 (del(11q), deletion 13q14 (del(13q), and trisomy 12). Most clinical FISH assays have high specificity and sensitivity, but the technique can detect a limited number of alterations per assay. Importantly, next-generation sequencing (NGS) techniques have become more readily available for clinical applications and are increasingly being used for screening not only mutations, but also copy number abnormalities in multiple genes and chromosomal regions of interest in hematologic malignancies. Here, we evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using a custom targeted NGS assay for detecting common prognostic chromosomal alterations in CLL and high-count monoclonal B-cell lymphocytosis (MBL), the precursor to CLL. Methods : We designed a SureSelect DNA targeted sequencing panel, covering all exons of 59 recurrently CLL mutated genes and additional amplicons across regions affected by clinically relevant CNAs. All CLL (N=534) and MBL (N=162) patients had pre-treatment peripheral blood mononuclear cells (PBMC) collected within two years of diagnosis. DNA was extracted in cases with purity >80% of CD5+/CD19+ cells. Clinical FISH data was available within 100 days of NGS in all untreated CLL and MBL cases. PatternCNV was used to detect clinically relevant CNAs in chromosomes 11, 12, 13 and 17. We performed a principal component analysis on the CNA calls, excluding chromosomes 11, 12, 13, and 17 to identify clusters of samples. Each cluster was then independently rerun with PatternCNV and the results from chromosomes 11, 12, 13, and 17 were extracted and further analyzed. We excluded samples with low tumor metrics identified by FISH (less than 20% of cells with del(17p), del(11q), trisomy 12 and del(13q)). Results: We sequenced a total of 696 patients of whom 162 were MBL and 534 were untreated CLL. The most commonly mutated genes were NOTCH1 (11.0%), TP53 (8.7%), SF3B1 (7.7%), ATM (4.1%), and CHD2 (3.8%). Based on CNA analyses from the NGS data, we identified 59 (9.1%) individuals with del(17p), 88 (13.4%) individuals with del(11q), 128 (20.0%) individuals with trisomy 12, and 329 (53.0%) individuals with del(13q). All 696 individuals had FISH panels conducted, with 39 (5.6%) individuals with del(17p), 68 (9.8%) individuals with (11q), 119 (17.1%) with trisomy 12, and 295 (42.4%) with del(13q). When we compared our CNA analyses with the FISH data, we found high concordance 95.0% for del(17p), 92.7% del(11p), 94.3% for trisomy 12, and 88.2% for del(13q). For del(17p) we found a sensitivity of 93.9%, specificity of 95.4%, PPV of 52.5%, and NPV of 99.7%. Del(11q) revealed a sensitivity of 88.1%, specificity of 94.0%, PPV of 59.1%, and NPV 98.8%. We found a sensitivity of 93.8%, specificity of 95.6%, PPV 82.0%, and NPV of 98.6% for trisomy 12 and for del(13q) we found a sensitivity of 92.6%, specificity of 90.9%, PPV of 91.7%, and NPV of 93.8%. We found lower PPVs in del(17p) and del(11q) likely due to lower prevalence of these chromosomal abnormalities. Conclusion: Here we show a high sensitivity, specificity, and NPV when comparing targeted sequencing with FISH. FISH panel testing is widely used in clinical practice to characterize highly prognostic chromosomal abnormalities in CLL. Comprehensive genetic profiling with NGS has become increasingly important in the work up of hematologic malignancies and provides additional prognostic and predictive information, including clinically relevant mutations such as TP53, SF3B1, and NOTCH1, tumor mutation load and mutations associated with resistance to chemo-immunotherapy and targeted therapies, such as BTK or BCL2 inhibitors, that FISH cannot offer. We show that NGS can infer clinically relevant CNA in cases without FISH testing while also providing additional clinically relevant information. Figure 1 Figure 1. Disclosures Cerhan: Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding; Genentech: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Genentech: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Acerta Pharma: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees.

Abstract Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies >0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.

Abstract Chronic lymphocytic leukemia (CLL) is a neoplastic disease of mature B-cells with a highly heterogeneous clinical course. While European ancestry (EA) populations present an increased incidence of CLL, African ancestry (AA) populations have a younger median age of onset, higher frequency of adverse prognostic factors, and inferior clinical outcomes. Despite the considerable effort to characterize the genetic landscape of CLL, AA are overwhelmingly underrepresented. Our hypothesis is that the clinical differences observed between AA and EA populations are, in part, explained by underlying genetic features. To address this imbalance, we identified 90 AA patients diagnosed with CLL, 64% of which were untreated at sample collection. RNA and DNA were extracted from CD5+/CD19+ clonal B-cells. We performed mRNA-seq and targeted sequencing in 59 recurrently mutated somatic CLL driver genes. Differentially expressed genes were identified using edgeR. Data was compared to our previously analyzed EA CLL cohort (N=445). The median age at diagnosis was 59 years for AA and 66 for EA and 74% of AA and 50% of EA had unmutated IGHV (u-IGHV) status. When evaluating the entire AA and EA cohorts, there was a significant increased frequency of mutations in TP53, SF3B1, and NFKBIE, identified in 29%, 24%, and 20% of AA CLLs, compared to 5%, 9%, and 9%, respectively, in EA CLLs (p<0.01). When exclusively evaluating the untreated and u-IGHV cases, AA CLLs showed greater proportion of TP53 (30% vs 12%; p=0.0145), NFKBIE (30% vs 15%; p=0.0492), BIRC3 (21% vs 10%; p=0.0697), and KRAS (15% vs 7%; p=0.1683). Furthermore, there was an increase in mutations targeting relevant molecular pathways, such as NF-κB (42% vs 15%) and MAPK (18% vs 8%). Upregulation MAPK pathway was also confirmed by mRNA-seq analysis in the AA u-IGHV CLLs. Because of the high prevalence of TP53 mutations in the AA cohort, we further evaluated differential gene expression in the DNA Damage/Telomere Stress-Induced Senescence pathway. AA CLLs presented a significant downregulation of multiple genes associated with genome stability and cellular DNA damage response - DDR (including TP53 and ATM), double strand break repair (H2AFX and RAD50), telomere maintenance (POT1 and ACD), and cell cycle regulation (RB1, CCNA1, and CCNE2) (FDR<0.05). DDR is responsible for DNA repair or induction of apoptosis, with its deficiency resulting in the accumulation of chromosomal aberrations, negatively impacting clinical outcome in CLL. Disparities in cancer are influenced by numerous factors that affect disease risk, screening and diagnosis, access to treatment, and survival. We identified an increased number of genomic alterations in the AA CLL cohort, primarily inducing activation of NF-κB and MAPK pathways and DDR impairment, with the increased frequency of mutations, notably in TP53 and BIRC3, expected to negatively impact prognosis. Citation Format: Cecilia Bonolo de Campos, Daniel R. O'Brien, Chantal E. McCabe, Huihuang Yan, Geffen Kleinstern, Zhiquan Wang, Laura A. Bruins, Cristine Allmer, Nicholas J. Boddicker, Charla R. Secreto, Aaron D. Norman, Shulan Tian, Kari G. Rabe, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Richard Furman, J Brice Weinberg, James R. Cerhan, Celine M. Vachon, Neil E. Kay, Susan L. Slager, Esteban Braggio. Characterization of underlying genomic features among African ancestry populations diagnosed with chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2209.

Abstract Immune dysfunction is a hallmark of CLL/MBL; ~25% individuals with newly diagnosed CLL have hypogammaglobulinemia at diagnosis. In this study of newly diagnosed CLL/MBL, we sought to a) investigate the association between serum immunoglobulins and specific mutations identified by next generation sequencing, and b) determine if serum immunoglobulin levels predict TFT, independent of the CLL-International Prognostic Index (CLL-IPI) and tumor mutational load (TML). We used the Mayo Clinic CLL Resource to identify newly diagnosed CLL/MBL individuals consented within 2 years of diagnosis. Baseline clinical characteristics and the CLL-IPI score was ascertained on all individuals. DNA was extracted from peripheral blood CD5+/CD19+ clonal B-cells; and the entire coding regions of 60 somatically recurring mutated CLL driver genes were enriched using a customized Agilent SureSelect gene panel and sequenced in Illumina Hiseq4000. Serum immunoglobulins were quantitated by radial immunodiffusion using Immunoplates. The relationship between serum immunoglobulins was assessed: a) with individual gene mutations (mutated vs. not); and b) using the TML score (using a value of 0, 1, and 2 or greater [2+] mutated genes). The impact of serum immunoglobulin levels on TFT was analyzed for all individuals. Of 356 individuals identified, 80 (22%) had MBL, median age was 63 years (28-87), 261 (73%) were male, and the CLL-IPI distribution was: low risk (n=149, 42%), intermediate risk (n=112, 32%), and high/very high risk (n=94, 26%). We observed 43%, 39%, and 18% individuals with 0, 1, or 2+ mutated genes, respectively. The most commonly mutated genes were NOTCH1 (13%), SF3B1 (13%), TP53 (8%), ATM (8%), BIRC3 (7%), and NFKBIE (7%). Using standard cutoffs, 135 (38%) individuals had low serum IgG, 73 (21%) had low serum IgA, and 154 (44%) had low serum IgM at diagnosis. There were no differences in individual gene mutation frequencies between individuals with low and normal serum IgG. In contrast, individuals with low serum IgA were more likely to have mutations in NOTCH1 (25% vs 10%) and ATM (14% vs 7%) compared to those with normal IgA, and individuals with low serum IgM were more likely to have mutations in NOTCH1 (19% vs 9%) and BIRC3 (11% vs 4%) compared to those with normal IgM (all p-values <0.05). There were no significant differences in individuals with low serum IgG based on TML scores of 0, 1, and 2+ (31%, 35%, 40%, respectively, p=0.28). However, individuals with low serum IgA and low serum IgM were more likely to have a higher TML (8%, 20%, 36% for IgA, p<0.0001; and 35%, 45%, 54% for IgM, p<0.0001, for TML scores of 0, 1, and 2+, respectively). After a median follow-up of 6.9 years, 157 individuals required CLL therapy. After adjusting for CLL-IPI and TML, low serum IgG, low serum IgA, and low serum IgM (hazard ratio [HR]: 1.8, 2.3, and 2.4, respectively, all p-values <0.05) were independently associated with a shorter TFT. Our study shows that individuals with CLL/MBL who harbor mutations in NOTCH1, ATM and BIRC3 are more likely to have low serum IgA and IgM at diagnosis. Serum IgA and IgM (but not serum IgG) levels decrease with an increase in the TML score. Finally, low serum immunoglobulins can predict TFT, independent of the CLL-IPI and TML, which has important implications for risk stratification in newly diagnosed CLL/MBL. Citation Format: Sameer A. Parikh, Cristine Allmer, Geffen Kleinstern, Nicholas J. Boddicker, Shulan Tian, Cecilia B. De Campos, Laura A. Bruins, Daniel R. o"brien, Brian F. Kabat, Kari G. Rabe, Aaron D. Norman, Huihuang Yan, Xing Li, Timothy G. Call, Wei Ding, Jose F. Leis, Saad J. Kenderian, Celine M. Vachon, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6466.

Abstract We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired PSMB5 mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. Implications: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.

Abstract Next‐generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL‐IPI) in newly diagnosed CLL and high‐count monoclonal B‐cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi‐stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time‐to‐first treatment (TTT), adjusted for CLL‐IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17‐1.39, P = 2.6 × 10 −8 ; c ‐statistic = 0.76). When stratified by CLL‐IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37‐1.72, P = 7.0 × 10 −14 ). Overall, 80% of low/intermediate CLL‐IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12‐2.07, P = .007; c ‐statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL‐IPI, especially among low/intermediate CLL‐IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.

Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p<0.05). We generated GLMs using an average of 92 markers (range 64-107) per compound, combining mRNA-sequencing expression with FISH and mutation data. The analysis proposed in the present study was validated through the unbiased selection of BCL2 among the subset of markers included in the GLM predicting sensitivity to venetoclax, a first-in-class orally bioavailable selective BCL2 inhibitor. Expression level of critical NF-kB and cell cycle genes, such as BIRC3, CKS1B, PAX5, NFKB2, and CCND2, were included in 60% of our predictive models. Mutations of DNA repair genes (ATM,TP53) were included in the GLMs of three epigenetic therapies, one histone deacetylase inhibitor and two BET inhibitors, associated to ex vivo resistance to the drugs. The presence of monosomy 13 was also a marker for ex vivo resistance for five epigenetic therapies, four HDAC inhibitors and one BET inhibitor. The three BET inhibitors, JQ1, CPI-0610, and OTX015, were among the compounds most accurately predicted by our integrative approach, with Spearman correlation values between 0.773-0.858. Overall, our models accurately predicted the ex vivo response for 16 (80%) of the compounds (r>0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r>0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

ABSTRACT Canine mammary neoplasms (CMNs) are the most frequent lesions and in female dogs. However, studies correlating pathological criteria with clinical evolution in female dogs with mammary neoplasms are scarce. The present study aims to present epidemiological, clinical-pathological and overall survival data to help establish the prognosis and understand the biological behavior of CMNs. A total of 1539 cases were included (85% malignant and 13% benign). Tumor size was an important prognostic factor and was associated with overall patient survival (P< 0.0001). Most dogs diagnosed with malignant neoplasms (83%) had initial clinical staging, although 17% had regional or distant metastases at the time of diagnosis and lower overall survival (P< 0.0001). Carcinoma in mixed tumor was the most frequent histological type and had a better prognosis. Solid carcinomas, micropapillary carcinomas and carcinosarcomas were considered histological types with aggressive biological behavior and were associated with a worse prognosis and lower overall survival (P< 0.0001).