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Richter's syndrome (RS) represents one of the foremost challenges in CLL management, and its pathogenesis remains largely undefined. We recently leveraged CRISPR-Cas9 in vivo gene editing to develop mouse models of RS by engineering multiplexed loss-of-function lesions typical of CLL (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) in early stem and progenitor cells [Lineage - Sca-1 + c-kit + (LSK)] from MDR-Cd19Cas9 donor mice. These animals express Cas9-GFP in a B-cell restricted fashion and the leukemogenic MDR lesion, which mimics del(13q) when the sgRNA-transduced LSK cells are transplanted in CD45.1 immunocompetent recipients. Through these methods, we observed not only development of CLL, but also transformation into RS, and even captured a stage where CLL and RS were co-existing in the same animal (CLL/RS). We hypothesized that the molecular events underlying RS development would be markedly distinct from those of CLL and performed transcriptome analysis of FACS-sorted CLL and/or RS cells (5 CLL, 4 CLL/RS, 10 RS) and normal B cell controls from 4 age-matched wild type MDR-Cd19Cas9 mice. We identified a unique transcriptional profile of RS (ANOVA, FDR We asked whether these oncogenic circuitries would be recapitulated in human RS. By correlating the differentially expressed genes in murine RS with those of 7 human RS cases (compared to matched CLL), we identified similar pathway dysregulations with >100 commonly altered genes including upregulated cell cycle regulators (CDK1, CCNA2) and downregulated signaling adapters (ITPKB, MAP3K9). To further dissect gene regulatory networks driving transformation in the mouse, we profiled one CLL and one RS case by single cell ATAC sequencing (scATAC-seq). Consistent with the RNA-seq profiles, we detected increased chromatin accessibility of MYC-family associated transcription factor motifs (MAX, MYCN), and reduced accessibility of the pro-inflammatory STAT2 motif in RS (-log10adjP>50). Functionally, decreased interferon gamma responses were confirmed by the reduced ability of RS cells to phosphorylate STAT1 and STAT3 at 5' and 15' after IFN-gamma stimulation, compared to CLL and normal B cells (Western Blot). To define the genetic landscape underlying these changes, we performed whole genome sequencing analysis, and identified loss of chr12 and chr16 as recurrent events in RS (6/8 cases) and CLL/RS (2/2), but not in CLL cases (0/5). Among the genes encoded by these chromosomes, we identified several epigenetic drivers (Dnmt3a, Crebbp, Setd3/4), MAP kinase family members (Map4k5, Mapk1), cytoskeletal regulators (Hcls1, Rhoj), and interferon family receptors (Ifnar1/2, Ifngr2), suggesting that broad epigenetic modifications together with loss of BCR and interferon signaling molecules represent key events of transforming disease. RS cases were also characterized by a significantly higher number of full chromosome amplifications or deletions (median=6; range: 2-9), as compared to CLL or CLL/RS (1; 0-5, P=0.0008), consistent with the high degree of genomic instability observed in human disease. Finally, we asked whether the observed changes would impact RS therapeutic vulnerabilities, and exposed 15 primary murine RS splenocyte samples to 20 drugs in vitro for 24 hours, followed by CellTiter-Glo assessment of cellular viability. We observed strong sensitivity to the BRD4 inhibitor JQ1 and the mTOR inhibitor everolimus (both reported to interfere with MYC signaling, P In conclusion, we define the evolutionary trajectories and therapeutic vulnerabilities of RS in a mouse model, with unique transcriptional, genetic, and epigenetic features, indicative of broad changes in MYC, IFN and BCR signaling pathways and remarkable similarities with human disease. In-depth analyses of BCR signaling and in vivo treatment studies are underway and will refine mechanistic insights into the biology of RS. Disclosures Davids: Surface Oncology: Research Funding; Eli Lilly and Company: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; Janssen: Consultancy; Verastem: Consultancy, Research Funding; Ascentage Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Merck: Consultancy; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; AbbVie: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; BeiGene: Consultancy. Letai: Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P We next assessed whether these cell models could predict responses of these combined abnormalities to BTK and PI3K inhibitors. We observed that HG3-del(11q) TP53MUT and HG3-TP53MUT cells showed partial response to ibrutinib and idelalisib, although the IC50 values were still higher than the ones observed in HG3WT clones, especially with idelalisib (27.4 and 20.6 vs. 1.8 uM, respectively). Nonetheless, we found that HG3-del(11q) TP53MUT cells were highly sensitive to novel preclinical drugs that have been shown to be effective in TP53 deficient cells such ATR inhibitors, with an IC50 value comparable to HG3WT cells (mean IC50 0.55 vs. 0.67 uM) (Fig 1C). In summary, we show that mutations in TP53 can appear in a subset of monoallelic del(11q) CLL cases, conferring a synergistic clonal advantage in vivo, and therefore a dismal clinical impact on the OS of this CLL subgroup. In addition, the biological basis of mutual exclusivity of biallelic ATM and TP53 alterations in CLL was assessed, underscoring the importance of the number of alleles affected by these alterations, and establishing novel pre-clinical models for the study of the biology and therapeutic response of concurrent genetic abnormalities in the disease. Funding: PI18/01500 FI19/00191 CD19/00222 *MQA CPC equal contr Disclosures No relevant conflicts of interest to declare.

Although we have gained a wealth of knowledge from large-scale DNA sequencing studies across blood cancers, we still know little about the functional interplay of the discovered putative drivers in the generation of chronic lymphocytic leukemia (CLL) and its transformation into Richter's syndrome (RS). We have previously observed that CRISPR-Cas9 in vivo B-cell editing of common CLL loss-of-function (LOF) lesions (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) can increase in vitro B cell fitness, but is not sufficient to sustain in vivo B cell survival after 12 months post-transplant. We therefore asked whether combinatorial introduction of mutations was required for CLL development. To this end, we generated transplant lines by in vitro engineering of early stem and progenitor cells (Lineage- Sca-1+ c-kit+ [LSK]) from MDR-/-Cd19-Cas9 donor mice (animals expressing Cas9-GFP in a B-cell restricted fashion and the leukemogenic homozygous MDR lesion, mimicking del(13q)) with pooled lentivirus expressing sgRNAs against the 6 genes of interest and the mCherry marker. Engineered LSKs were then re-transplanted into sub-lethally irradiated immune-competent CD45.1 or immune-deficient NSG recipients (n=35/strain). Parallel control cohorts of equal size were generated by transducing LSKs with a pool of 6 non-targeting sgRNAs. Disease development (B220+CD5+Igk+ cells) was assessed by flow cytometric analysis of bi-monthly peripheral bleeds, starting at 4 months post-transplant, and flow cytometry/IHC were utilized to classify tumors at euthanasia. Analysis of PCR-based targeted NGS of peripheral blood edited tumor cells (GFP+mCherry+) was performed utilizing CRISPResso software to assess presence of the 6 LOF mutations. We observed incidence of circulating CLL in 28/35 (80%) CD45.1 and 27/35 (77%) NSG mice, whereas only 5/35 (14%) CD45.1 and 4/35 (11.4%) NSG from the non-targeting control cohort developed CLL-like disease (P To determine the genetic composition of the 55 leukemias/lymphomas (n=22, 11 and 22, with patterns A, B and C), we interrogated the LOF mutational burden at euthanasia. We observed a median number of 4 LOF mutations (range:1-6), and a high overall frequency of Trp53 lesions (58%). The other 5 drivers were less prevalent (Mga: 26%; Chd2: 21%; Samhd1: 17%; Birc3 and Atm: 13%). Trp53 mutations were predominantly clonal (≥90% indels, P In conclusion, we demonstrate that combinatorial in vivo modeling of CLL-LOF mutations leads not only to CLL development, but also to RS, thus establishing a faithful framework for analysis of genetic and microenvironmental determinants of disease transformation. We are now interrogating genome-wide mutational patterns and clonal architecture of CLL vs. RS, while analyzing their microenvironmental composition. These novel models provide a unique platform to discern critical insights into RS pathogenesis and to discover RS-specific therapeutic vulnerabilities. Disclosures Clement: Edilytics: Current Employment, Current equity holder in private company. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company.

Amongst the novel putative drivers identified by large-scale sequencing studies of chronic lymphocytic leukemia (CLL) is the ribosomal protein RPS15. Mutated in 5.3% of CLL, it co-occurs with heterozygous TP53 alterations in 36% of RPS15-mutated samples. Mutation of this mediator of ribosome maturation and translation is associated with poor disease prognosis and enriched in cohorts with del(17p) and relapsed CLL, suggesting a role in disease progression and therapeutic resistance. However, the impact of RPS15 mutation on B cell function and CLL development, in the presence or absence of TP53 mutation, has yet to be characterized. To this end, we developed overexpression HG3 CLL cell lines modeling four common RPS15 mutations (G134R, H137Y, S138F, and S139F) and a conditional knock-in mouse model of the S138F mutation with and without heterozygous Trp53 deletion (generated by crossing Rps15 and Trp53 mutant mice with Cd19-Cre mice). To characterize the impact of RPS15 mutation on transcription, we performed RNA-sequencing on splenic B cells from 3-month-old Rps15WT, Rps15Het and Rps15Hom mice (3 per cohort). We identified 255 and 670 upregulated and 596 and 777 downregulated genes in the Rps15MT vs Rps15WT mice (Rps15Het and Rps15Hom, respectively; log2FC>0.5, p1, nominal p-value To evaluate whether RPS15 mutant proteins incorporate into ribosomes, we performed polysome profiling of the HG3 lines. All overexpressed RPS15-WT and MT proteins were observed to integrate into the small ribosomal subunit and mature ribosomes, potentially impacting translation. Next, ribosome profiling of HG3 RPS15-WT and S138F cells revealed 2,334 genes with differential translation efficiency (TE) between RPS15-S138F vs WT cells and 2,425 genes between RPS15-S138F vs WT in TP53 knock-out cells (log2FC>0.5, p To determine whether Rps15 mutation can drive CLL-like disease, we engineered 6 novel mouse lines with B cell restricted expression of alterations through crossing with CD19-Cre mice: Rps15WT, Rps15Het, and Rps15Hom mutant mice alone or co-expressing Trp53 deletion. We detected circulating CLL-like (B220+CD5+) cells in 5 of 30 (17%) Rps15Het mice by 20 months of age, but not in 30 age-matched Rps15WT mice. We also detected CLL-like cells in 6 of 30 (20%) Trp53+/- mice by 17 months, indicating that Trp53 deletion alone can induce CLL-like disease. Interestingly, we found CLL-like cells in 2 of 30 Rps15Het/Trp53+/- mice as early as 15 months of age. The cohorts of Rps15Hom and Rps15Hom/Trp53+/- mice, however, have been monitored for 18 months of age with no disease occurrences, indicating that a double dosage of Rps15 mutation may be detrimental to disease formation. Altogether, Rps15 heterozygous mutation can drive CLL development in mice, and our early data hint that co-mutation with Trp53 may shorten the latency of CLL-like disease. Overall, RPS15 mutant protein can incorporate into the ribosome and induce changes in mRNA translation, resulting in MYC activation predominantly in the context of TP53 loss. Our mouse studies indicate that mut-Rps15 drives CLL development, with a more aggressive disease course when combined with Trp53 deletion. Our results collectively suggest that RPS15 and TP53 co-mutation drives CLL development through translational dysregulation and MYC-mediated signaling. Disclosures Neuberg: Pharmacyclics: Research Funding; Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company. Getz:Broad Institute: Patents & Royalties: MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, POLYSOLVER and TensorQTL; Pharmacyclics: Research Funding; IBM: Research Funding; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences (>70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: 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. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.

Background: Chromosome 14q32 rearrangements involving the immunoglobulin heavy chain gene (IGH) affect less than 5% of chronic lymphocytic leukemia (CLL) patients. Their clinical course is aggressive and the outcome, worse than other CLL subtypes (Cavazzini et al, 2008; Gerrie et al, 2012). However, the biology of CLL showing IGH rearrangements (CLL-IGHR) is not completely defined. The identification of novel recurrent mutations in CLL by next generation-sequencing (NGS) has offered a more comprehensive view into the genomic landscape of the disease and improved the prognostication of CLL. Thus, mutational analysis might be especially useful in those patients with uncertain prognosis, such as those carrying IGH rearrangements. Aim: To analyze the mutational profile of CLL-IGHR patients by targeted NGS in order to improve our understanding of the genetic underpinnings of this subgroup. Methods: The study was based on 899 CLL patients, well characterized at cytogenetic, biological and clinical level, forty-two of them (4.7%) showing IGH rearrangements. Targeted NGS was performed in 231 CLL samples: 117 with 13q deletion, 27 with 11q deletion, 26 trisomy 12, 42 showing IGH rearrangements and the remaining 19 without any cytogenetic alteration. CD19+ B cells were isolated and DNA extracted. SureSelectQXT targeted enrichment technology and a custom-designed panel (MiSeq, Illumina), including 54 CLL-related and recurrent mutated genes, was carried out. The panel yielded 100x or greater coverage on 97% of the genomic regions of interest and the mean coverage obtained was 600x. Mutations were detected down to 3% allele frequency. Results: The mutational analysis of CLL-IGHR patients identified a total of 72 mutations in 32 genes. Seventy-one percent of patients (30/42) harbored at least one mutation. The most frequently mutated genes in this cohort were NOTCH1 (28.6%), POT1 (14.3%), TP53 (9.5%), SF3B1 (7%), BRAF (7%), EGR2 (7%), IGLL5 (7%) and MGA (7%), followed by BCL2, HIST1H1E and FBXW7 (4.8%), uncommonly mutated genes in CLL at these frequencies (Table 1). In fact, mutations in NOTCH1, BRAF, EGR2, BCL2, HIST1H1E and FBXW7 were significantly associated with CLL-IGHR patients (p=0.013, p=0.003, p=0.021, p=0.038, p=0.038 and p=0.021 respectively). In terms of time to the first therapy (TFT), CLL-IGHR had an intermediate-negative impact (median TFT=24 months) compared to the presence of cytogenetic alterations associated with good prognosis such as 13q deletions (median TFT>120 months; p A total of 17 out of 42 CLL-IGHR patients (40.5%) carried the t(14;18). Interestingly, patients with t(14;18) were characterized by: 1) A lower mutation frequency (average of mutations/patient=1.05) than the rest of rearrangements with unknown partners (average=2.16; p=0.039), and 2) The presence of mutations in BCL2 (11%) and HIST1H1E (11%). By contrast, CLL-IGHR without BCL2 rearrangement showed mutations in POT1 (20%), TP53 (16%), SF3B1 (12%) and BRAF (12%). Moreover, t(14;18) was significantly associated with good prognosis markers such as the mutated status of the variable region of the immunoglobulin genes (IGHV-M) (p=0.002). However, there was no significant difference in terms of TFT between patients with t(14;18) and patients with other IGH rearrangements (p=0.27). Conclusions: CLL patients with IGH rearrangements showed: i. A high gene mutation frequency; ii. A distinct mutational profile, with recurrent mutations in POT1, EGR2, BRAF, IGLL5 and MGA genes; iii. An adverse clinical outcome refined by the negative effect of genetic mutations. iv. Patients with t(14;18) presented a lower mutation frequency than the rest of rearrangements, carrying mutations in BCL2 and HIST1H1E, and associated with good-prognosis markers such as IGHV-M. Funding:PI15/01471; CIBERONC CB16/12/00233; FEHH-Janssen(MHS); JCyL(MQÁ) Disclosures Mateos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies. To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition). Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients. PI15/01471 SA085U16 JCyL-MQ FEHH-MH Disclosures García-Tuñón: Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Clonal evolution is considered as a hallmark of progression in chronic lymphocytic leukemia (CLL). Next-generation sequencing technologies have expanded our knowledge of genetic abnormalities in CLL and enabled to describe marked clonal changes. The acquisition of driver mutations accompanied by selectively neutral passenger changes may be essential to understand the transformation from diagnosis to later more aggressive stages. However, the role of genetic mutations and clonal evolution during the clinical progression prior any therapy is still largely unknown. Longitudinal studies analyzing CLL patients repeatedly before intervening treatment are currently scarce. Patients and methods: We examined the exomes from 35 CLL patients in 2 time-points. Two groups of patients were characterized: (i)patients with progression (n=26) in which we analyzed samples taken from an early stable stage (inactive disease) and during clinical progression (active disease), but before treatment (median of time to first treatment=2.7 years); (ii)patients without progression with a stable inactive disease until last follow-up (n=9) (median follow-up=5.25 years). We also compared patients that gained new cytogenetic aberration detected by FISH in the 2nd time-point with those who did not. Sequencing libraries were prepared using TruSeq Exome Enrichment and sequenced by Illumina HiSeq1000 (84X). Somatic mutation calling was performed by a standardized bioinformatics pipeline. Thereafter, driver mutations were identified using the Cancer Genome Interpreter (https://www.cancergenomeinterpreter.org), a novel tool that identifies variants that are already validated as oncogenic and predicts the effect of the mutations of unknown significance. Results: We identified 397 somatic mutations in 364 different genes, ranging from 4 to 26 mutations per patient. Among them, 58 driver mutations were identified, being SF3B1 (6/35, 17.1%), TP53 (4/35, 11.4%) and NOTCH1 (4/35, 11.4%) the most common mutated genes. Comparing progressive vs. stable group, CLL patients with clinical progression showed a higher intra-tumoral heterogeneity than cases without progression (median of somatic mutations=14[4-26] vs. 9[5-14]). Comparing both tumoral time-points in the same patient, we identified a total of 11 acquired driver mutations and 7 mutations increasing its allele frequency in more than double in the 2nd time-point respect to the 1st one. All of them were detected in patients with clinical progression. Interestingly, TP53 and BIRC3 exhibited recurrently acquired mutations (detected each one in 2 cases). Three driver mutations in cancer genes not yet known for CLL (DHX9, GNAQ and HDAC2) were also acquired. Within CLL progressive patients (n=26), we observed clonal evolution characterized by acquired cytogenetic aberration in 9 cases. In patients with progression but no cytogenetic aberration gained at the 2nd moment (n=17), we detected that almost half of them (7/17) showed clonal evolution by acquired or doubled driver mutations. In the remaining patients with clinical progression but without any clonal evolution (n=10), 6 cases showed a driver mutation of CLL genes associated with bad prognosis (SF3B1, TP53, NOTCH1 or RPS15) already at first time-point. In the stable group (n=9), none acquired or doubled mutation was detected. However, clonal evolution characterized by acquired cytogenetic aberration was observed in 4/9 stable patients: two of them acquired 13q- whereas the other two acquired 11q-. Within stable patients without clonal evolution (n=5), we detected one case with a driver mutation in SF3B1 already at 1st time-point (follow-up=5 years). Conclusion: Clonal evolution represents a central feature of tumor progression in CLL. Our data show that the disease is evolving during time even in stable patients without any clinical signs of disease activity. In progressive patients, the disease evolution is accompanied by new appearance or accumulation of driver mutations and cytogenetic aberrations. Moreover, progressive patients that showed less or no changes during time bore typical CLL drivers at the first time-point. Funding: Seventh Framework Programme (NGS-PTL/2012-2015/no.306242); Ministry of Education, Youth and Sports (2013-2015, no.7E13008; CEITEC 2020 (LQ1601)); AZV-MZ-CR 15-31834A-4/2015 and TACR (TEO2000058/2014-2019); PI15/01471; Junta de Castilla y León (MHS). Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 1776 Array-based sequence capture (Roche NimbleGen) followed by next-generation sequencing (Roche GS FLX Titanium sequencing platform) was used to analyze genetic variations in 93 genes relevant in CLL and two chromosomal regions: 13q14.3 and 17p13.1. CD19+ cells from 4 patients with CLL and 4 patients with other hematological malignancies (used as controls) were studied. A custom-made data analysis pipeline was used to annotate detected variants, including known single-nucleotide polymorphisms (SNPs), amino acid consequences, genomic location and miRNA binding sites. The enrichment assay followed by NGS allowed the detection of over 1600 variations/sample (median 1721, range 1618–1823). All putative variants were first compared with published single nucleotide polymorphism (SNP) data (dbSNP build 130) and most of the variants detected were identified as known SNPs. Overall, 10% of variants detected in each sample were variations not previously described. Interestingly, a 4bp insertion/deletion polymorphism (rs2307842) in the 3′UTR of HSP90B1, target site for miR-223, was detected. There is an increasing evidence suggesting that SNPs in the 3′UTR targeted by miRNAs (known as miRSNPs) are associated with diseases by affecting gene expression. We hypothesized that this ‘GACT’ deletion disrupts the binding site for miR-223 thereby increasing the translation of HSP90B1 and we confirmed that miR-223 regulates HSP90B1 expression by 3′UTR reporter assays. The relative luciferase activity of the construct with wild-type 3′UTR (WT-3′UTR) was significantly repressed by 31% following miR-223 transfection (p Downregulation of miR-223 has prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients, and no target has been proposed or validated for miR-223 in CLL until date. Thus, this work provides novel information about how the downregulation of miR-223 can be determining the poor outcome of CLL patients, maybe through upregulation of HSP90B1 expression. Disclosures: No relevant conflicts of interest to declare.

The mutational status of the immunoglobuin heavy variable (IGHV) genes is an undisputable strong prognostic factor that subdivides patients with chronic lymphocytic leukemia (CLL) into 2 subgroups, i.e. IGHV-unmutated CLL (U-CLL) and IGHV-mutated CLL (M-CLL). U-CLL and M-CLL have distinct landscapes of genomic aberrations as well as distinct prognosis, since U-CLL is considerably more aggressive than M-CLL. That said, there is considerable clinical heterogeneity among M-CLL patients, ranging from patients without need of treatment to patients requiring early therapeutic intervention, indicating the need to further refine prognosis in this subgroup. In recent years, it has become evident that the prognostic impact of genomic aberrations may differ depending on IGHV gene mutational status. Hence, defining genomic aberrations with prognostic impact in M-CLL patients may help identifying patients with an predicted unfavorable prognosis within this subgroup, with obvious implications for management of follow up and therapy choice. To study the clinical impact of recurrent gene mutations in relation to IGHV gene mutational status, we collected a large, multi-center cohort including 4,674 patients with CLL [median age at diagnosis, 64.5 years; male/female, n=2,962 (63%)/n=1,712 (37%); Binet stage A (n=3,369, 74%), B (n=827, 18%), and C (n=387, 8%); IGHV-mutated (M-CLL, n=2,498, 56%) and IGHV-unmutated (U-CLL, n=1,927, 44%); isolated del(13q) (n=1,868, 41%), trisomy 12 (n=571, 13%), del(11q) (n=503, 11%), and del(17p) (n=249, 5.5%); treated (n=2,745, 59%) and untreated (n=1,929, 41%)] and performed next-generation sequencing (NGS) and/or Sanger sequencing of 9 genes (BIRC3, EGR2, NFKBIE, MYD88, NOTCH1, POT1, SF3B1, TP53, and XPO1) on pre-treatment samples. Overall, pathogenic mutations in any of these genes were detected in 1720/4674 patients (36.8%, using a variant allele frequency cutoff of 5% for NGS), while the remaining patients were wildtype; 2 mutations were observed in 361 patients (7.7%) and 3 or more mutations in 58 patients (1.2%). The mutation frequency for the individual genes was: TP53 (10.4%, including TP53 mutations and/or del(17p)), NOTCH1 (10.1%, 3'UTR mutations not included), SF3B1 (9.3%), XPO1 (3.9%), POT1 (3.8%), NFKBIE (3.7%), BIRC3 (3.0%), EGR2 (2.5%) and MYD88 (2.5%; Figure 1A). Except for MYD88, gene mutations in each of the investigated genes were associated with significantly shorter time-to-first-treatment (TTFT) in univariate analysis. In multivariate analysis of Binet stage A patients (n=3,369; including all genes, IGHV gene mutational status, age at diagnosis and gender), SF3B1 (Hazard Ratio (HR) 1.79; p In summary, different spectra of genetic mutations independently predicted short TTFT in M-CLL and U-CLL, respectively, with SF3B1 mutations as the only aberration found to be significant predictor of shorter time to first treatment in both subgroups. Importantly, mutations within several genes (i.e. SF3B1, NOTCH1, XPO1 and NFKBIE) identified patients in the M-CLL subgroup with a high-risk profile; conversely, TP53 mutations did not affect TTFT in this subgroup. On these grounds, we suggest to include analysis of recurrent gene mutations to identify high-risk patients within the M-CLL subgroup. Figure 1 Figure 1. Disclosures Brieghel: AstraZeneca: Consultancy. Rossi: Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Scarfo: Astra Zeneca: Honoraria; Abbvie: Honoraria; Janssen: Honoraria, Other: Travel grants. Mattsson: Gilead: Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Martinez-Lopez: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Serna: AbbVie, AstraZeneca, Beigene, Gilead, GSK, Janssen, Jazzpharma, Novartis, Roche: Consultancy; ABBVIE, ASTRAZENECA,ROCHE: Research Funding; AbbVie, AstraZeneca, Roche: Speakers Bureau. Hernández Rivas: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Smedby: Jansen-Cilag: Other: part of a research collaboration between Karolinska Institutet and Janssen Pharmaceutica NV for which Karolinska Institutet has received grant support. Bullinger: Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Hexal: Consultancy; Janssen: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Menarini: Consultancy; Novartis: Consultancy, Honoraria; Amgen: Honoraria; Astellas: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Research Funding. Bosch: TAKEDA: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel. Terol: BMS: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Roche: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hospital Clinico Valencia: Current Employment. Cuneo: AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau. Gaidano: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemann: Novo Nordisk Foundation: Research Funding; CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding. Ghia: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sunesis: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria.