B cell receptor antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. To investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within eight weeks in diseased mice. Furthermore, we tested whether mutations augmenting B cell signaling influence the course of CLL development and its severity. The Phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Regulation of B Cell Receptor Signaling and Its Therapeutic Relevance in Aggressive B-Cell Lymphomas

The proliferation and survival signals emanating from the B-cell receptor (BCR) constitute a crucial aspect of mature lymphocyte’s life. Dysregulated BCR signaling is considered a potent contributor to tumor survival in different subtypes of B cell non-Hodgkin lymphomas (B-NHLs). In the last decade, emergence of BCR-associated kinases as rational therapeutic targets has led to the development and approval of several small molecule inhibitors targeting either Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (SYK), or phosphatidylinositol 3 kinase (PI3K), offering alternative treatment options to standard chemoimmunotherapy, and making some of these drugs valuable assets in the anti-lymphoma armamentarium. Despite their initial effectiveness, these precision medicine strategies are limited by primary resistance in aggressive B-cell lymphoma like diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), especially in the case of first generation BTK inhibitors. In these patients, BCR-targeting drugs often fail to produce durable responses, and nearly all cases eventually progress with a dismal outcome, due to secondary resistance. This review will discuss our current understanding of the role of antigen-dependent and antigen-independent BCR signaling in DLBCL and MCL and will cover both approved inhibitors and investigational molecules being evaluated in early preclinical studies. We will discuss how the mechanisms of action of these molecules, and their off/on-target effects can influence their effectiveness and lead to toxicity, and how our actual knowledge supports the development of more specific inhibitors and new, rationally based, combination therapies, for the management of MCL and DLBCL patients.

The Determinants of B Cell Receptor Signaling as Prototype Molecular Biomarkers of Leukemia

Advanced genome-wide association studies (GWAS) identified several transforming mutations in susceptible loci which are recognized as valuable prognostic markers in chronic lymphocytic leukemia (CLL) and B cell lymphoma (BCL). Alongside, robust genetic manipulations facilitated the generation of preclinical mouse models to validate mutations associated with poor prognosis and refractory B cell malignancies. Taken together, these studies identified new prognostic markers that could achieve characteristics of precision biomarkers for molecular diagnosis. On the contrary, the idea of augmented B cell antigen receptor (BCR) signaling as a transforming cue has somewhat receded despite the efficacy of Btk and Syk inhibitors. Recent studies from several research groups pointed out that acquired mutations in BCR components serve as faithful biomarkers, which become important for precision diagnostics and therapy, due to their relevant role in augmented BCR signaling and CLL pathogenesis. For example, we showed that expression of a single point mutated immunoglobulin light chain (LC) recombined through the variable gene segment IGLV3-21, named IGLV3-21R110, marks severe CLL cases. In this perspective, we summarize the molecular mechanisms fine-tuning B cell transformation, focusing on immunoglobulin point mutations and recurrent mutations in tumor suppressors. We present a stochastic model for gain-of-autonomous BCR signaling and subsequent neoplastic transformation. Of note, additional mutational analyses on immunoglobulin heavy chain (HC) derived from non-subset #2 CLL IGLV3-21R110 cases endorses our perspective. Altogether, we propose a model of malignant transformation in which the augmented BCR signaling creates a conducive platform for the appearance of transforming mutations.

Final Results of a Phase 1/2 Study of SYK Inhibitor Entospletinib in Combination with Obinutuzumab in Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL)

Therapeutic resistance and intolerance of Bruton tyrosine kinase (BTK) inhibitors is an emerging need in CLL. SYK is integral to the activation of BTK and the B-cell receptor (BCR) signaling cascade and is overexpressed in CLL. We have shown that BAFF-mediated SYK activation triggered BCR signaling and rendered protection of CLL cells from spontaneous apoptosis in vitro. Single agent small molecule SYK inhibitor entospletinib was efficacious in treatment of patients with R/R CLL. Here we report final results of a single arm, open label, investigator-initiated phase 1/2 clinical trial which evaluated safety and efficacy of entospletinib in combination with obinutuzumab, a glycoengineered monoclonal anti-CD20 antibody, in patients with R/R CLL (NCT03010358). Patients enrolled in the Phase 1 dose-escalation portion of the trial included adults with CLL or non-Hodgkin lymphoma (Phase 1 part only) after ≥1 prior therapy. Patients were enrolled at 2 dose levels to receive entospletinib 200 or 400 mg twice-daily orally according to 3+3 design. The primary endpoint for the phase 1 portion of the study was to determine the RP2D of the combination. All patients received single agent entospletinib as part of a 7-day run-in. Thereafter, patients received entospletinib on days 1-28 of each 28-day cycle continuously, and obinutuzumab intravenously in standard doses for 6 cycles. Once the RP2D was determined, a phase 2 study enrolled patients with R/R CLL only, where complete response (CR) was the primary endpoint. A total of 24 patients (22 CLL, 2 follicular lymphoma) were enrolled. Twelve patients were enrolled in the phase 1 part of the study. The phase 2 part of the study included 17 patients with CLL. Of 6 patients who received entospletinib 200 mg on the Phase 1 part of the study, one patient experienced a DLT (grade 3 asymptomatic AST/ALT abnormalities) attributed to entospletinib. No DLTs were observed among the six patients who received entospletinib 400 mg. Thus, entospletinib 400 mg twice-daily was determined to be the RP2D in combination with obinutuzumab. Efficacy of entospletinib+obinutuzumab was analyzed in the 21 patients with CLL, of which 17 received entospletinib at RP2D (400 mg twice daily). Patients with CLL had a median age of 66 years. Thirteen patients (62%) had TP53 aberration (n=9), complex karyotype (n=6), or NOTCH1 or SF3B1 mutation. The median number of prior therapies was two (range, 1-6). Seven patients had received prior ibrutinib (4 patients discontinued due to intolerance and 2 due to progression). Median follow-up was 31 months. Among the 21 efficacy-evaluable participants with CLL, the ORR was 67% (95%CI, 43-85%). Three patients (14%, 95%CI 3-36%) achieved a CR, and 11 patients (53%) had a partial response (PR). patients with confirmed CR had undetectable MRD in the bone marrow. Median event-free survival was 27.5 months (95%CI: 16 months-NR), treatment duration - 31 months (95%CI: 27-40; Figure). Thirteen patients with high-risk CLL had an ORR of 54% (5 PRs and 2 CRs). Among the eight patients who had previously received kinase inhibitors, ORR was 62.5% (all PRs). Treatment-related adverse events were reported in 96% of patients (Table). Grade 3 or higher AEs occurred in 65%. Neutropenia (43.5%; including 4 patients [17%] who had transient grade 4 neutropenia attributed to obinutuzumab) was the most common grade ≥3 hematologic toxicity. The median onset of neutropenia was 7 days after the first obinutuzumab infusion, median duration was 28 days. Growth factor support was not required and grade ≥3 infection occurred in only 1 patient. Only one patient on study discontinued therapy due to adverse events (recurrent AST/ALT abnormalities which resolved upon cessation of entospletinib). Pharmacodynamic analysis demonstrated that treatment with entospletinib led to rapid downmodulation of pSTAT3 and the anti-apoptotic protein MCL1 in CLL cells. Furthermore, six months of combination therapy was accompanied by a reduction in IFNγ secretion in CD4 + T-cells and a reversal of exhausted phenotype, as evidenced by downregulation of PD-1. Thus, the combination of entospletinib and obinutuzumab shows an acceptable safety profile. Efficacy of this combination (EFS 27.5 months in predominantly high-risk population ) compares favorably with chlorambucil/obinutuzumab in R/R CLL (13 months), thus warranting continued exploration of the regimen. Figure 1 Figure 1. Disclosures Danilov: Genentech: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding. Spurgeon: Bristol Myers Squibb: Other: Institution: Research Grant/Funding; BeiGene: Other: Institution: Research Grant/Funding; AstraZeneca: Other: Institution: Research Grant/Funding; Acerta Pharma: Other: Institution: Research Grant/Funding; Pharmacyclics: Consultancy; Janssen: Consultancy, Other: Institution: Research Grant/Funding; Genentech: Consultancy, Other: Institution: Research Grant/Funding; Karyopharm: Consultancy; Velos Bio: Consultancy, Other: Institution: Research Grant/Funding; Gilead Sciences: Other: Institution: Research Grant/Funding; Ionis: Other: Institution: Research Grant/Funding; Merck & Co., Inc.: Other: Institution: Research Grant/Funding; Fred Hutchinson Cancer Research Center: Other: Data Safety Monitoring Board. Kittai: Abbvie: Consultancy; Bristol-Meyers Squibb: Consultancy; Janssen: Consultancy.

MYD88 L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation. In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 L265P or MYD88 WT, alone or in association with CD79B Y196F. In all three cell lines, the co-expression of MYD88 L265P and CD79B Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88L265P in CD79BY196F-associated proximal BCR signaling in DLBCL. To elucidate the potential role of the DOCK8 adapter in MYD88 L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 WT or MYD88 L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 L265P/DOCK8 signals in comparison to MYD88 WT/DOCK8 signals (p<.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 L265P rather than MYD88 WT (p<.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88L265P and CD79B genetic alterations. We next analyzed the consequences of MYD88 L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88L265P/CD79BY196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 WT/CD79B WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88L265P/CD79BY196F DLBCL cell lines but not in lines with MYD88 WT/CD79B WT, highlighting the specific role of DOCK8 in MYD88 L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88L265P/CD79BY196F, but not MYD88WT/CD79BWT, DLBCLs (p<.004, HBL1 and p<.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88L265P/CD79BY196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88L265P/CD79BY196F mutations. Disclosures Shipp: AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Bayer: Other: Institution: Research Grant/Funding; Abbvie: Other: Institution: Research Grant/Funding.

B Cell Receptor Signaling Drives APOBEC3 Expression Via Direct Enhancer Regulation in Chronic Lymphocytic Leukemia B Cells

Introduction: CLL is the most common leukemia in the U.S. and characterized by constitutively activated BCR signaling pathway, which has a crucial role both in normal B cell development and B cell malignancies. The biological events controlled by BCR signaling in CLL are not fully understood. Active BCR signaling is mediated through activation of the downstream kinase Bruton tyrosine kinase (BTK), which has become a key therapeutic target to inhibit BCR signaling for the treatment of B cell malignancies. We reasoned that blood samples from CLL patients before and after Bruton's tyrosine kinase inhibitors (BTKi) treatment would provide a valuable resource in the study of BCR modulation of epigenetic machinery in leukemic B cells. Methods: We obtained blood samples from CLL patients before and after BTKi ibrutinib treatment and used them to study BCR signaling regulated genes (n = 8 patients, after one-year of continuous ibrutinib treatment). Gene expression profile of CLL B cells from patients before and after one-year ibrutinib treatment was analyzed by mRNA-seq seq. Genome wide Histone H3K4me1, H3K27ac, and H3K4me3 profile was determined by CUT&tag. Chromatin accessibility was determined by ATAC-seq. Putative enhancers were deleted by CRISPR-Cas9. Results: Notably BTKi treatment led to the reduction of expression in genes associated with single strand DNA deamination (Fig. 1A) The BTKi regulated genes involved in this process mainly contains the APOBEC3 family genes (APOBEC3C, APOBEC3D APOBEC3F, APOBEC3G, APOBEC3H), and their expression levels showed a consistent reduction in CLL B cells from ibrutinib treated patients (Fig. 1B). We then confirmed the reduction of APOBEC3 levels by western blot in CLL B cells from four patients before and after one-year of continuous ibrutinib treatment (Fig. 1C). We hypothesized that BCR signaling regulates APOBEC3 expression by modifying the local chromatin around the APOBEC3 gene cluster and performed CUT&Tag to map the histone marks including H3K4me1, H3K4me3, H3K27ac and ATAC-seq. This approach permitted us to examine the chromatin accessibility of the leukemic cells from CLL patients before and then after one-year of continuous ibrutinib treatment. We found that BTKi treatment caused reductions of H3K4me1, H3K27ac, and chromatin accessibility at these regions in ibrutinib treated patients ( 7 of the 8 samples tested), however, there was no change of the promoter marker H3K4me3 (Fig. 1D), which indicated that BTKi treatment leads to APOBEC3 genes expression change via the regulation of their enhancer regulation (APOBEC3 enhancers, AEs). Based on the enrichment of H3K4me1, H3K27ac and chromatin accessibility, AE regions of the ibrutinib native samples contain three active enhancer modules, we designated these modules as AE1, AE2, and AE3 (Fig. 1D). To assess the functional activity of these enhancers on the expression of APOBEC3 genes, we investigated the consequence of deletion of each one of these AEs in the MEC1 cell line by CRISPR-Cas9. PCR analysis showed very robust deletion of AE1, AE2, and AE3 (Fig. 1E). Both deletion of AE1 or AE2 reduced the expression of APOBEC3 genes, while AE3 deletion suppressed the expression of APBEC3C, APOBEC3D, APOBEC3F and APOBEC3G, but not APOBEC3H, which is in closest proximity to AE3 (Fig. 1F, G). Together, we identified the BCR signaling dependent enhancers that regulate APOBEC3 expression. Since APOBEC3 deaminates ssDNA, we reasoned that APOBEC3 in CLL B cells may also contribute to replication stress and DNA instability. We found that MEC1 cells have a high level of spontaneous DNA damage in the S phase cells (Fig. 1H). which is associated with replication stress. However, AE2 deleted MEC cells showed decreased γH2Ax in the S phase cells (Fig. 1H). Edu/PI assay showed that MEC1 cells had a fraction of S phase cells with low Edu incorporation during S phase, also indicating DNA replication stress (Fig. 1I); however, AE2 deletion greatly increased Edu incorporation (Fig. 1I). Taken together, these data suggest that increased expression of APOBEC3 may be involved in DNA replication stress and drives genomic instability in malignant B cells. Conclusion: We demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in CLL B cells, implicating BCR signaling as a potential regulator of leukemic genomic instability. Figure 1 Figure 1. Disclosures 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: AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Agios Pharm: 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; Sunesis: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; 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; MEI Pharma: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Analyses of the Kinetics and Phenotype of Multiple Intraclonal CXCR4/CD5 B Cell Subsets Suggest Differences in Life Cycle Transitioning in CLL

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease so that defining the dynamic features of the clone and its intraclonal subpopulations are essential to understand disease pathogenesis and to develop novel, effective therapies. For instance, because cell division is linked with new mutations, the ability to preferentially select cells that recently divided allows studying the subpopulation(s) most likely responsible for disease progression and resistance to therapies. The intraclonal kinetics of CLL B cells have been studied in clonal subgroups defined by reciprocal surface levels of CXCR4 and CD5. In that model, three fractions are identified: recently divided "proliferative" (PF; CXCR4 DimCD5 Bright); "intermediate" (IF; CXCR4 IntCD5 Int) and "resting" (RF; CXCR4 BrightCD5 Dim). Here, we have expanded the examination of subpopulations differing for time since last division ("age"). Unmanipulated CLL cells studied ex vivo from 10 patients who drank 2H 2O for 4 weeks were sorted by the relative densities of CXCR4 and CD5 to isolate the formerly identified PF, IF and RF as well as two fractions not previously characterized, "Double Dim" (DDF: CXCR4 DimCD5 Dim) and "Double Bright" (DBF; CXCR4 BrightCD5 Bright). For each fraction, the amount of deuterium incorporated into cellular DNA in vivo was measured. Consistently, the PF contained significantly higher levels of 2H-labeled DNA and higher calculated cell division rates when compared with the RF and IF. Interestingly, the DDF also contained significantly more 2H-labeled DNA compared to the RF; in contrast, the DBF resembled more closely the RF fraction. The overall 2H-incorporation gradient was: PF>DDF>IF>DBF>RF. In CLL, BCR signaling is fundamental, with the amount of membrane (m) IgM associating with signaling competence and disease aggressiveness. Additionally, when engaged independently, mIgM and mIgD can lead to different signaling sequelae. Therefore, we analyzed the 5 subpopulations for the densities of mIgM and mIgD. This showed a distribution similar to that of 2H-DNA incorporation: for IgM: PF=DDF>IF=DBF=RF, and for IgD: PF>DDF>IF=DBF>RF. Accordingly, we next measured 2H-DNA in subpopulations with low, intermediate and high levels of IgM and IgD. This revealed a direct correlation between IG densities and in vivo DNA synthesis, consistent with intraclonal subpopulations with high IGs having divided more recently than those with low IGs. However, these findings are not in line with cell division being primarily initiated by BCR engagement since that would lower mIgM levels. Therefore, we tested if engagement of TLR9 would affect mIG densities on CLL cells. After stimulation of 32 CLL clones with CpG+IL15, anti-IgM+IL4, anti-IgD+IL4, or anti-IgM-IgD+IL4, there was a significant increase in mIGs only after CpG+IL15 activation; each anti-IG stimulation led to downregulation of mIGs. Finally, we questioned the subclonal responsiveness to BTK inhibition in vivo. CLL samples taken from the same patients, before and during ibrutinib treatment, displayed intraclonal changes in mIG densities and cell size, the latter a marker of cellular and metabolic activation also linked with CLL in vivo birth rates. Ibrutinib treatment normalized mIgM and mIgD intraclonal densities and lead to an overall cell size decrease with larger, 2H-enriched and higher mIG density cells being more affected (PF>DDF>IF>DBF>RF). Collectively, these findings suggest that the most recently born cells enter the circulation as the PF from which they transition to either lower CD5 (DDF) or higher CXCR4 (IF and DBF) phenotypes. Each eventually converge as the RF. Moreover, since mIG densities on the more recently divided populations (PF and DDF) are high, the data imply that successful cell division is not solely a consequence of BCR engagement; the involvement of the TLR pathways, concomitantly or in series with BCR signaling, is more consistent with the higher mIG levels. Finally, ibrutinib treatment appears to preferentially target more recently divided cells with high mIG levels. Disclosures Allen: Alexion: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees.

Dissecting Richter's Syndrome in a Multiplexed CRISPR-Based Mouse Model Reveals Key Changes in MYC, Interferon and BCR Signaling Underlying Transformation

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<0.1), characterized by upregulation of pathways involved in cell survival and proliferation (E2F/MYC targets, G2-M checkpoint, mitotic spindle). In contrast, genes involved in interferon gamma response, JAK-STAT and BCR signaling were predominantly downregulated. 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<0.0001), and to CDK inhibitors (e.g. the CDK4/6 inhibitor palbociclib, P=0.0007), modest activity of the JAK1/2 inhibitor ruxolitinib (P=0.05), and minimal, if any, response to ibrutinib, venetoclax and fludarabine. 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.

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas. Disclosures Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

Transcriptome Based Classification Model and Co-Expression Networks Analysis Identify CXCR4alt Subtype and Blnkaf in B-Cell Precursor Acute Lymphoblastic Leukemia

Background B-cell precursor acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of acute leukemia with stage-specific phenotypes and cytogenetic features. Although the research on the molecular profile of B-ALL benefits diagnosis and risk stratification, the idiographic leukemogenesis beyond the transcriptome remains unknown. Genomic lesions in B-ALL frequently involve genes belonging to transcription factors, such as TCF3, EBF1, PAX5, and IKZF1. The investigation of dysregulated transcriptional networks behind various B-ALL subtypes may help unravel the specific process of leukemogenesis. Methods A random forest model was trained on a well-defined molecular subtype B-ALL cohort (n = 504) to improve the molecular classification. The subtype-specific transcriptional network was constructed by weighted correlation network analysis (WGCNA) once the B-ALL subtypes were genetically determined by the random forest model. Additionally, alternative splicing analysis from RNA-seq was emphasized since aberrant splicing events could lead to abnormalities in transcription factors or tumor suppressor genes. Results The random forest model performs well for the classification of most B-ALL subtypes (Figure 1A). It also benefits the classification of Ph-like B-ALL, which displays a gene expression profile similar to BCR-ABL1 B-ALL, as it achieves 100% accuracy on well-known Ph-like cases characterized by ABL-class gene fusions, PAX5-JAK2, EBF1-PDGFRB, and IGH-EPOR. We successfully separated a candidate molecular subtype characterized by CXCR4 alteration (CXCR4alt) for the first time, through our novel classification model (Figure 1B). This newly identified CXCR4alt subtype accounts for 2% of B-ALL cases (11/504), characterized by CXCR4 C-terminal mutation R334X or FLNA overexpression. Both C-terminal mutation and upregulated FLNA contribute to delayed CXCR4 receptor internalization, enhanced CXCL12-CXCR4 signaling, and then continuously activates the downstream MAPK pathway. It is further supported by the high expression of the two oncogenic MAPK signaling pathway genes KIAA1549 and KIAA1549L from the co-expression network of CXCR4alt in these cases. Transcriptional co-expression networks constructed by WGCNA and network hub genes for most B-ALL subtypes also help to elucidate the mechanism of leukemogenesis (Figure 2). We identified an alternative first exon of BLNK (BLNKaf) that leads to loss of function as a shared event in specific subtypes, such as BCR-ABL1, BCR-ABL1-like, and PAX5alt; while in pre-BCR signaling positive subtypes, such as TCF3-PBX1 and MEF2D-r, only express normal BLNK transcripts. Discussion By comprehensive transcriptome-based classification model and co-expression networks analysis, we identified a novel defined CXCR4alt subtype with an incidence of 2% in B-ALL. We also observed that BLNKaf might supply a practical marker for monitoring pre-BCR signaling. Our report emphasizes the role of transcriptome-based machine learning and WGCNA in mining the molecular mechanism of B-ALL. The molecular pathogenesis and clinical significance of these newly identified molecular subtypes and molecular abnormalities are worthy of further investigation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.