IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

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Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽

10.1182/blood-2020-136172 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hanyin Wang ◽

Shulan Tian ◽

Qing Zhao ◽

Wendy Blumenschein ◽

Jennifer H. Yearley ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

Expression Profiles ◽

Lymphocytic Leukemia ◽

B Cell Activation ◽

Advisory Committees ◽

Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

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Chronic Lymphocytic Leukemia B Cells Display IgM and IgD Isotype-Restricted Features That Affect Association with Co-Receptors, BCR Signaling, and Leukemic B-Cell Growth In Vivo

Blood ◽

10.1182/blood-2018-99-119989 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3124-3124

Author(s):

Andrea Nicola Mazzarello ◽

Marcus Dühren-von Minden ◽

Eva Gentner ◽

Palash Chandra Maity ◽

Gerardo Ferrer ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cell Size ◽

Metabolic Activity ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Bcr Signaling

Abstract The leukemic cells in patients with chronic lymphocytic leukemia (CLL) are highly dependent on B-cell receptor (BCR) mediated signaling. Despite this and the fact that >90% of CLL clones co-express IgM and IgD, the composition and molecular mechanisms regulating BCR signaling regarding the two isotypes and the co-receptors with which they associate is lacking. Here we have addressed these issues. First, using Imaging Flow Cytometry, we evaluated BCR organization on the surface membrane of CLL cells from 11 patients who had participated in a 2H2O-labeling study that determined in vivoCLL B-cell birth rates (BR). We found that in all cases mIgM resided in more and larger surface clusters than mIgD. Also, a statistically significant, direct correlation was observed for IgM density and in vivoCLL-cell BR, with patients exhibiting more recently-divided cells having the highest expression of IgM. This was not the case for IgD. BCR signaling requires co-receptors that can co-localize differently with the two isotypes. Thus, we tested co-localization of stimulatory (CD20) and inhibitory (CD22) co-receptors with mIgM and mIgD, using the proximity ligation assay technique that discriminates 10 to 40 nm distances. Higher IgM:CD20 and lower IgD:CD20 co-localization ratios directly associated with in vivo BR. Conversely, patients whose CLL B cells showed greater IgM to CD22 co-localization ratios had lower BRs. Thus, association of IgM with stimulatory versus inhibitory co-receptors correlated with positive or negative regulation of CLL growth in vivo. Next, we questioned the extent that the observed differences in BCR organization affected the entire clone by measuring a marker of single cell metabolic activity - cell size. IgM and BR associated with entire clonal populations that were skewed toward larger, more active cells. Similarly, high BR CLLs displayed an increased mitochondrial maximal respiration and glycolytic activity and capacity, based on measurements of oxygen consumption rate and extracellular acidification rate, respectively. Since our findings supported a link between IgM- but not IgD-BCRs, growth rate in vivoand clonal metabolic activity, we questioned whether intrinsic, constitutive CLL BCR autonomous signaling differed for these two isotypes. To address this, we examined the signaling capacities of CLL-derived BCRs expressed as IgM or IgD isotypes, while maintaining the original IGHV-D-J and IGLV-J rearrangements. We used B cells that do not express endogenous BCR-related molecules but do express an inducible ERT2- SLP-65 fusion protein which enables examining Ca++influx. All BCRs expressed as IgM effectively mobilized Ca++ without need for an external ligand, indicating autonomous signaling. In contrast, BCRs expressed as IgD did not signal autonomously but required crosslinking with anti-BCR. Thus, only mIgM BCRs naturally transduce a signal in the absence of antigen. To determine the extent that BCR signaling influences clonal activity and in vivoBR, we compared cell size of CLL B cells taken from patients before and after 4 weeks of treatment with the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib (iBTK). Ibrutinib had a strong treatment effect on cell activity, reducing overall cell size in 10/11 patients. A comparison of single cell areas for patients with lower (BR = 0.54%) and higher (BR = 1.42%) BRs showed an overall reduction of the median cell size for both cases. Thus, iBTK treatment leads to an equilibration of the cell size profile among the cases differing in BR, indicating that ibrutinib acts proportionally more potently on more metabolically active CLL B cells. Likewise, these findings are consistent with BCR signaling, transduced through BTK, being responsible for the increased cellular activity of aggressive CLL clones. In conclusion, increased mIgM density and proximity of mIgM to stimulatory receptors is linked to greater metabolic activity clones and increased rate of proliferationin vivo. Conversely, proximity of mIgM to inhibitory receptors has the opposite correlations.Moreover, only mIgM carries out autonomous signaling, providing another biologic trait linking all these features. Thus, our data support a tight, isotype-dependent regulation of BCR signaling and its consequences for CLL B cells. Further understanding these mechanisms should help generate novel therapies to modify the quality of BCR-transduced signaling and thus cell fate. Disclosures Barrientos: Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: 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. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

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Tecfidera Modulates Activation of Malignant B-Cells: Correlative Analysis from a Phase 1 Clinical Trial in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-119292 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3143-3143

Author(s):

Christina Wu ◽

Emanuela M. Ghia ◽

Fitzgerald Lao ◽

Minya Pu ◽

Karen Messer ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

Phase 1 ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Phase 1 Clinical Trial ◽

Bcr Signaling

Abstract Tecfidera (Dimethylfumarate, DMF) is an immunomodulatory drug currently approved in the United States for the treatment of patients with multiple sclerosis (MS). DMF formulations have also been utilized for the treatment of patients with psoriasis. The ability of DMF to Inhibit autoreactive T-lymphocytes may also be applicable to malignant T- and B- lymphocytes. Indeed, preclinical studies by multiple groups have highlighted its potential efficacy as a treatment for patients with various lymphoid cancers, though inhibition of STAT, NF-KB, Wnt signaling pathways, and glycolysis. This information provided rationale to conduct a phase 1 clinical trial of DMF as a treatment for patients with relapsed chronic lymphocytic leukemia (CLL). In this study we report the correlative assessment of the initial patients who received DMF. Patients with relapsed or refractory CLL received DMF 120 mg PO BID (based on preclinical modeling with primary CLL cells in vitro and in vivo and 50% lower than the standard DMF dose for patients with MS). Treatment was planned for a short duration of 2 months to evaluate tolerability and effects on CLL cell signaling and tolerability of the drug by patients with CLL. Peripheral blood leukemic cell were isolated and analyzed by RNAseq and phosphoprotein immunoblotting at times just prior to, and 6 hours immediately following drug administration. Consistent with preclinical results (Brennan et al, PLOS one 2015; Selman et al, Science Trans Med 2018), STAT1 phosphorylation at Y701 was decreased 6 hours following the initial dose, compared to high baseline levels. Differential expression analyses for RNAseq of total RNA isolated from peripheral blood CLL cells weere performed using limma models, which compared pretreatment versus post-treatment at a specific time point. Functional annotations of these genes revealed a significant enrichment related to B-cell activation, B-cell receptor (BCR) signaling, B-cell differentiation, and immune signaling generally. Specifically, several known CLL therapy targets including SYK, LYN, and BTK were significantly downregulated. Gene-set-enrichment analysis (GSEA) revealed that the "CLL-BCR gene signature" previously described by Herishanu et al. (Blood 2011) was consistently down-regulated after DMF treatment in samples from different time points (C1D1 post, C1D8 and C2D1) compared to pretreatment samples (FDR q of 0.24, 0.01 and 0.04, respectively). In conclusion, this is the first report of the clinical use of DMF for patients with CLL in which inhibition of leukemic cell activation has been demonstrated. The inhibitory effect of DMF on leukemic cell gene expression, in particular its antagonism of BCR signaling, is valuable information for the development of novel combination strategies for CLL or other B-cell malignancies. Disclosures Kipps: F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Choi:Gilead: Speakers Bureau; Genentech: Speakers Bureau; Rigel: Consultancy; AbbVie, Inc: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau.

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Chronic Lymphocytic Leukemia Patients with IGHV Genes Carrying Only Silent Mutations Have A Longer Time From Diagnosis to Initial Therapy Than Patients Expressing B-Cell Receptors with No Somatic Mutations

Blood ◽

10.1182/blood.v118.21.288.288 ◽

2011 ◽

Vol 118 (21) ◽

pp. 288-288 ◽

Cited By ~ 1

Author(s):

Matthew Kaufman ◽

Xiao J. Yan ◽

Emanuela M. Ghia ◽

Donna Neuberg ◽

Laura Z. Rassenti ◽

...

Keyword(s):

Amino Acid ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Antigen Binding ◽

Advisory Committees ◽

Acid Structure ◽

Amino Acid Structure

Abstract Abstract 288 BACKGROUND. Chronic lymphocytic leukemia (CLL) patients with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHV genes (U-CLL). It has been proposed that this difference reflects the fact that IGHV mutations alter the structure of the B-cell antigen receptor (BCR) such that it no longer binds stimulatory (auto) antigens and therefore cannot deliver trophic signals to the leukemic cells. For this theory to be correct, only replacement (R) mutations and in particular non-conservative R mutations that would more likely alter amino structure of the IGHV/D/J rearrangements would have relevance. Silent (S) mutations by definition do not change amino acid structure and could not alter antigen binding. We sought to investigate this hypothesis by analyzing the types (S, conservative R, non-conservative R) and distribution of mutations that occur in IGHVs of M-CLL clones and then comparing the time to first therapy (TTFT) in patients with different IGHV features. This analysis expanded an initial study of 1569 CLL cases in the US to include 1858 patients from Europe for a total of 3427 cases. METHODS. Using IGMT software and tools, we analyzed the rearranged IGHV sequences of 3427 cases and characterized their mutations in several respects: first, if IGHV mutations altered amino acid structure (S vs. R); second, if mutations occurred in CDRs (antigen binding domains) or FRs (scaffolds of the BCR); third, if R mutations were conservative or non-conservative as determined by charge, hydropathy, and size. TTFT for patients was examined with various combinations of the above parameters. Differences in TTFT were estimated by the method of Kaplan and Meier and assessed using the log rank test. RESULTS. First, TTFT was compared for 4 groups of patients with the following mutation profiles: no mutations; only S mutations (median 1 per sample); only R mutations (median 1 per sample); and mixed S and R mutations (median 16 per sample). These 4 categories were significantly different (P<0.0001), with the median TTFT being 2.0 yrs for no mutations; 2.6 yrs for R only; 2.8 yrs for S only; and 7.3 yrs for mixed. All comparisons used patients with no mutations (n=1234) as the reference group. We identified statistically longer TTFT (2.8 vs 2.0 yrs; P=0.04) in patients with only S mutations (n=62). Patients with only R mutations (n=218) also had superior outcomes (2.6 yrs vs. 2.0 yrs; P=0.008). There was no statistical significance in TTFT between patients with all S vs. all R mutations (2.8 yrs vs. 2.6 yrs; P=0.71). Because exploring the relevance of BCR structural change on TTFT required us to focus on a small subset of patients with only silent IGHV mutations, our sample size was small. Therefore, we expanded the group to include IGHVs with R mutations limited to the FR region (S + R in FR only; n=241) since these mutations would be unlikely to have a major impact on BCR structure, particularly sparing the binding site. When compared to patients with no mutations, this group demonstrated a significantly longer TTFT (2.8 yrs vs. 2.0 yrs, P=0.0002). This finding was upheld when not permitting any non-conservative R mutations in this group, leaving only patients with S plus conservative R mutations in FRs only (n=124); TTFT in this case was 2.8 yrs vs. 2.0 yrs, respectively (P=0.0006). CONCLUSIONS. Our findings show that patients with only S mutations have better outcomes than patients with no mutations, suggesting that a change in BCR structure that could lead to a loss of antigen binding is an unlikely reason for improved clinical course. This is further supported by the finding that the combination of only S plus conservative R mutations located solely in FRs, which probably would not result in major BCR structural changes and therefore antigen binding, is associated with a lengthened TTFT. Therefore, we suggest that the currently accepted paradigm to explain the enhanced survival of M-CLL patients needs to be re-evaluated. Disclosures: Brown: Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

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Reduced Expression of the CD94/NKG2C NK Cell Receptor in Chronic Lymphocytic Leukemia (CLL) and CLL-like Monoclonal B-Cell Lymphocytosis (MBL)

Blood ◽

10.1182/blood-2019-128055 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5457-5457

Author(s):

Anna Puiggros ◽

Gonzalo Blanco ◽

Aura Muntasell ◽

María Rodríguez-Rivera ◽

Lara Nonell ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Lymphocytic Leukemia ◽

Study Cohort ◽

Cell Compartment ◽

Advisory Committees

Background. Dysregulated NK-cell responses have been reported in chronic lymphocytic leukemia (CLL) patients, but little is known about the NK cell compartment in CLL-like monoclonal B cell lymphocytosis (MBL). Human cytomegalovirus (HCMV) infection induces an adaptive reconfiguration of the NK cell compartment characterized by the differentiation and persistent expansion of a subset displaying the CD94/NKG2C NK receptor (NKR). Moreover, a deletion of the NKG2C (KLRC2) gene has been reported to modulate the magnitude of the NK cell repertoire redistribution. Little is known about the expression of NKG2C in CLL and MBL patients. Aims. To analyse the distribution of NKR, with special attention to NKG2C, in MBL and CLL patients, assessing the relation of the NK cell immunophenotype with clinical features. Methods. The study cohort included 61 patients, 24 were diagnosed with clinical MBL and 37 were treatment-naïve CLL (32/37 Binet A). The expression of NKG2C, NKG2A, ILT2 (LIR1, LILRB1), CD161, CD57 and KIRs (identified with a cocktail of monoclonal antibodies) was assessed by flow cytometry in peripheral blood NK cells. The NKG2C (KLRC2) genotype was analysed in a larger representative MBL/CLL cohort (n=135). Results. The proportions of NK cells were reduced in CLL patients compared to MBL (median 5.5% vs. 10%; P=0.003), whereas their absolute numbers were increased (median 0.85x109/L vs. 0.57x109/L; P=0.002). No significant differences between MBL and CLL were detected regarding the distribution of the different NKR: NKG2C (median: 2.7 vs. 5.9%, respectively), NKG2A (31.4 vs. 30.8%), ILT2 (18.0 vs.15.8%), KIRs (54.4 vs. 52.7%), CD161 (16.1 vs. 16.4%) and CD57 (40.4 vs. 38.9%). Though a reduced NKG2C expression was noticed in both entities, it was specially marked in patients with a greater (>30x109 cells/L) lymphocytosis (1.4 vs. 7.7%, P=0.016). The proportions of NKG2C+ NK cells in HCMV+ patients (85%, 47/55) as compared to HCMV- individuals were not significantly different (6.3% vs. 2.9%, respectively). HCMV+ patients showed a significantly lower NKG2C expression when compared with two independent age-matched cohorts of HCMV+ non-CLL/-MBL individuals, including 43 non-metastatic breast cancer patients (4.2% vs. 15.3% , P<0.001); and 30 renal transplantation donors (4.2% vs.12.4% in P=0.028). The frequencies of NKG2C+/+ (56%), NKG2C+/del (37%) and NKG2Cdel/del (7%) genotypes were comparable to those previously defined in healthy blood donors. Moreover, cases with very low (<2%) or undetectable NKG2C expression were found in NKG2Cdel/del patients (100%, 6/6), but also among NKG2C+/- (45%, 10/22) and NKG2C+/+ (12%, 3/26) genotypes. Conclusions. 1. MBL and CLL exhibited low proportions of NKG2C+ NK cells. This immunophenotype was particularly evident in CLL patients with increased lymphocytosis and could not be explained by differences in HCMV seropositivity, NKG2C zygosity nor age. 2. Additional studies are required to define the mechanism(s) and putative implications of the reduced NKG2C expression in these lymphoproliferative disorders. Acknowledgements. PI11/1621; PI15/437; 2017/SGR437; Fundació La Caixa; Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Abrisqueta:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. Bosch:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

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B-Cell Receptor Driven MALT1 Activity Regulates MYC Signaling in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.611.611 ◽

2016 ◽

Vol 128 (22) ◽

pp. 611-611

Author(s):

Beiying Dai ◽

Michael Grau ◽

Mélanie Juilland ◽

Pavel Klener ◽

Elisabeth Höring ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

Cell Receptor ◽

B Cell Receptor ◽

Advisory Committees ◽

Mantle Cell ◽

Bcr Signaling

Abstract Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor prognosis. Recent studies revealed the importance of constitutive B-cell receptor (BCR) signaling in maintaining survival of a subset of MCLs. MALT1 is an essential component of the CARD11-BCL10-MALT1 (CBM) complex that links BCR signaling to the nuclear factor kappa-B (NF-κB) pathway. Additionally, MALT1 functions as a protease that cleaves various substrates to promote proliferation and survival. However, its role in the molecular pathogenesis of MCL is unknown. To elucidate the functional role of MALT1 in the biology of MCL, we determined its proteolytic activity in primary MCL cells and in MCL derived cell lines. A large fraction of MCLs displayed constitutive activity of MALT1. This MALT1 activity is driven by constitutive BCR signaling, as we were able to show that RNA interference-mediated knockdown of central components of the BCR cascade abolished MALT1 activity. To gain insights into the functional significance of MALT1 in MCL, we knocked down its expression by different MALT1 shRNAs. Transduction of these shRNAs induced cytotoxicity in models that are characterized by constitutive MALT1 activity, whereas no effect on survival was observable in MCLs without MALT1 activation. To determine if this MALT1 addiction translates into an in vivosetting, we knocked down MALT1 in mouse MCL models and detected a significant inhibition of tumor growth. This indicates that MALT1-activated MCLs are dependent on the function of MALT1. These results were confirmed as pharmacologic inhibition of MALT1 significantly reduced cell viability in MALT1-activated MCLs, implying that MALT1 inhibition might represent a promising therapeutic strategy for MCL patients. To understand which biologic processes are regulated by MALT1 in MCL, we profiled gene expression changes at different time points following MALT1 inhibition. An unbiased gene set enrichment analysis identified various previously described MYC gene expression signatures to be among the top downregulated signatures, suggesting that MALT1 regulates MYC expression. MYC downregulation following MALT1 inhibition or MALT1 knockdown was confirmed at the protein level and various analyses revealed that MALT1 regulates MYC expression posttranslationally by preventing its proteasomal degradation. These results were further confirmed in primary mouse splenocytes, indicating that this novel molecular mechanism of regulating MYC expression is not restricted to MCL. To confirm that MYC is indeed expressed in primary MCLs, we determined MYC expression in 234 primary MCL samples by immunohistochemistry. These analyses revealed that 75 samples (32.1%) displayed an intermediate and 55 samples (23.5%) a high MYC positivity, suggesting that MYC is expressed in a substantial number of primary MCLs. As common alterations such as MYC high-levelamplifications and translocations determined by FISH occurred extremely rarely in our primary MCL samples (1% of samples), it is conceivable that BCR-driven MALT1 signaling is the predominant mechanism of MYC upregulation in MCL. In summary, we report for the first time that a substantial fraction of MCLs is addicted to constitutive MALT1 signaling. Thus, MCLs can be divided based on their MALT1 activation status into two distinct subgroups. We further identified a novel regulatory mechanism of MYC expression by MALT1. Thus, our study provides a strong mechanistic rationale to investigate the therapeutic efficacy in targeting the MALT1-MYC axis in MCL patients. Disclosures Trneny: Janssen Research & Development: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreyling:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau.

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Expression of the Tigit/CD226/CD155 Receptors/Ligand System in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128308 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5454-5454 ◽

Cited By ~ 2

Author(s):

Francesca Arruga ◽

Giulia Guerra ◽

Denis Baev ◽

Catherine Hoofd ◽

Marta Coscia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees

Introduction: T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a surface receptor mainly expressed by CD8+, regulatory T lymphocytes and natural killer (NK) cells, but not by normal B cells. It performs as an inhibitory immune checkpoint, activated through binding of CD155. TIGIT competes with CD226 for CD155 binding, resulting in opposite outcomes: while CD226 enhances cytotoxicity of T lymphocytes and NK cells, TIGIT exerts immunosuppressive effects. Whether TIGIT engagement triggers an alternative signaling cascade, or whether it simply prevents CD226 activation, remains an open point. Tumor-infiltrating T lymphocytes generally express high levels of the molecule, together with the other checkpoint inhibitor PD-1. On this basis, antagonist antibodies targeting TIGIT are under evaluation to restore immunity and treat cancer patients, alone or in various combinations. Chronic lymphocytic leukemia (CLL), the most common adult leukemia, is characterized by a highly heterogeneous clinical outcome. Several molecular markers can help in stratifying patients, including the presence or absence of somatic mutations in B cell receptor, cytogenetic aberrations and single gene mutations. Interestingly, CLL cells express several T cell specific antigens, including CD5. A previous report indicates that, in CLL, TIGIT is expressed by circulating CD4+T cells, increasing during disease progression, while nothing is known about its expression on CLL cells. Aim:This work was undertaken with the aim of studying expression of the TIGIT/CD226/CD155 axis in CLL. Methods:We assembled a cohort of 101 primary CLL samples (40% females, mean age of 61). All patients were either untreated or had not received treatment in the 6 months prior to analysis. PBMC samples were tested for expression of TIGIT, CD155 and CD226 in both T and B subsets. A multiparametric flow cytometry strategy was designed, combining anti-TIGIT, anti-CD155 and anti-CD226 antibodies with a panel of B- (anti-CD19, anti-CD5, anti-CD38, anti-CD49d and anti-CD73) and T-mono/NK specific (anti-CD3, anti-CD8, anti-CD4, anti-CD14 and anti-CD56) markers. The number of TIGIT molecules on leukemic cells was estimated by interpolating values of mean fluorescence intensity (MFI) of each sample with that of PE-Quantibrite beads. Results:CLL cells heterogeneously express surface TIGIT, ranging from 0.2 to 81% (mean value 20%, median 10%, SEM ±2.145). The estimated number of molecules per cell was in the range of 32.5-3571 (mean 1140, median 841.1, SEM ±83.6). Expression of TIGIT was independent of gender or age at diagnosis and there was no correlation between TIGIT levels and lymphocyte counts in peripheral blood. In contrast, in this cohort of untreated patients, we observed a significantly lower TIGIT expression in samples with advanced disease (RAI III-IV) compared to early stages (RAI 0-I). Accordingly, low TIGIT associated with unmutated (UM) IGHVgenes and with an unfavorable FISH profile (trisomy 12, deletion 17 and deletion 11 vs. deletion 13 or normal karyotype). Lower, although not significant, TIGIT levels were observed in NOTCH1-mutated CLL samples (n=11) compared to counterpart (n=89). Looking at the T cell population, we observed overall higher TIGIT levels in the CD8+vs CD4+subset (mean %TIGIT+cells in CD8+56.7±1.8 vs 27.2±1.3 in CD4+). In line with reported observations, we found a modest but significant increase of TIGIT+T cells in advanced stage CLLs, at variance with what observed on the leukemic B cell side. Accordingly, we observed higher percentages of TIGIT+/CD4+cells in CLL samples carrying UM IGHVgenes. CD226 and CD155 were more homogeneously expressed in all subsets without significant differences, both in CLL and T cell components. Conclusions: This work shows that CLL cells express the immunomodulatory molecule TIGIT, particularly in the early stages of the disease in untreated patients. While further studies are needed to characterize its functional implications as well as treatment effect on TIGIT expression, it is tempting to speculate that TIGIT expression by CLL cells may serve to trigger an immunosuppressive behavior in these cells, which is no longer needed when the disease becomes advanced. This observation represents a starting point for future studies investigating the role of TIGIT in CLL and hints to a possible use of anti-TIGIT antibodies to target different cellular components of the disease. Disclosures Hoofd: iTeos Therapeutics: Employment. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Gaidano:Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Furman:Acerta Pharma: Consultancy; Beigene: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Deaglio:VelosBio Inc.: Research Funding; Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding.

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Correlations Between Ofatumumab Exposure and Treatment Outcomes for Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Frontline Ofatumumab, Fludarabine, and Cyclophosphamide Chemoimmunotherapy

Blood ◽

10.1182/blood.v118.21.1793.1793 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1793-1793 ◽

Cited By ~ 1

Author(s):

William G. Wierda ◽

Roxanne C. Jewell ◽

Thomas J. Kipps ◽

Jan Dürig ◽

Laimonas Griskevicius ◽

...

Keyword(s):

Monoclonal Antibody ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Mass ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Multivariable Analyses

Abstract Abstract 1793 Introduction: Results: Seven pts (4 male) with a medianLittle is known about the pharmacokinetics (PK) and pharmacodynamics of CD20 monoclonal antibody (mAb) with chemotherapy in patients (pts) with CLL. Ofatumumab (O) is a human mAb targeting a membrane-proximal small-loop epitope on CD20 and mediates efficient complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Safety and efficacy of O at 2 dose levels in combination with fludarabine and cyclophosphamide (FC) were evaluated in previously untreated pts with CLL. Relationship between O PK, baseline characteristics, and clinical outcomes were studied. Pts and Methods: Pts with active CLL were randomized to O 500 mg (n=31) or 1000 mg (n=30) on Day 1 with F 25 mg/m2 and C 250 mg/m2 on Days 2–4 (Course 1) or Days 1–3 (Courses 2–6) every 4 weeks for 6 courses. O dose at Course 1 was 300 mg for both groups. Response (1996 NCI-WG criteria) was assessed by an Independent Review Committee up to 3 months after last course. Serial blood samples were collected at Courses 1 and 6 for noncompartmental PK analyses; pre- and post-infusion samples were collected at other courses. Relationship between PK parameters and baseline pt characteristics and disease factors was evaluated by univariable and multivariable analyses. Associations between PK and complete response (CR), overall response (OR), or progression-free survival (PFS) were explored using univariable regression analyses. Results: 22/31 (71%; 500 mg) and 19/30 (63%; 1000 mg) of pts received all 6 O doses; 2 pts at 1000 mg did not receive FC for Course 6. CR (primary endpoint) rates of 32% and 50% and OR rates of 77% and 73% were observed in the 500 mg and 1000 mg groups, respectively. O PK parameters are summarized (Table). O PK at Dose 6 appeared proportional to dose. Factors associated with PK in multivariable analyses are shown (Table). The factor most associated with PK at Dose 1 was sex, with higher Cmax/AUC and lower CL and Vss/longer t½ in women vs. men. PK at Dose 6 was not consistently associated with any factor tested. Median Cmax and Cmin values were similar at first dose between pts who had CR, partial response (PR)/nPR, and stable disease (SD)/progressive disease (PD; Figure); at later doses, median Cmax and Cmin values appeared different between CR and the other groups, although number of subjects decreased over time, especially in the SD/PD group. Based on univariable analyses, higher Cmax and Cmin at Dose 3 and higher Cmax and AUC at Dose 6 were associated with increased likelihood of CR (P<.05); higher Cmin before Dose 6 was associated with increased likelihood of OR and longer PFS. Conclusions: PK of O in combination with FC appeared proportional to dose after repeated dosing. Higher concentrations at Doses 3 and 6 were associated with CR. O PK was similar at first dose and different at later doses between patients who had CR, PR/nPR, and SD/PD, suggesting that response to O-FC treatment leads to clearance differences due to decreased B-cell mass and thus concentration differences with continued dosing. Further analyses of associations between disease-related factors, PK, and treatment response will be performed at study completion. Disclosures: Wierda: GlaxoSmithKline: Research Funding; Genentech: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Merck: Consultancy; Abbott: Research Funding. Off Label Use: Ofatumumab is an anti-CD20 monoclonal antibody approved for the treatment of fludarabine- and alemtuzumab-refractory chronic lymphocytic leukemia, and is currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma), as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Jewell:GlaxoSmithKline: Employment. Kipps:Gilead Sciences: Consultancy, Research Funding; GSK: Research Funding; Genentech: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Abbot Industries: Research Funding; Celgene: Consultancy, Research Funding; Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Dürig:Santaris Pharma: Consultancy, Research Funding; GSK: Speakers Bureau; Roche: Speakers Bureau; Celgene: Research Funding. Stilgenbauer:Genmab: Research Funding; GSK: Consultancy, Honoraria, Research Funding. Smolej:Roche: Honoraria, Travel Grants; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Genzyme: Honoraria, Travel Grants. Hernandez-Ilizaliturri:Genmab: Research Funding; Celgene: Honoraria; Amgen: Research Funding. Fang:PharStat: Employment; GSK: Consultancy; Gilead Sciences: Consultancy; Pharmasset Inc: Consultancy. Gorczyca:GlaxoSmithKline: Employment. Chan:GlaxoSmithKline: Employment. Gupta:GlaxoSmithKline: Employment. Lisby:Genmab A/S: Employment.

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Polygenic Risk Score and Risk of Chronic Lymphocytic Leukemia, Monoclonal B-Cell Lymphocytosis (MBL), and MBL Subtypes

Blood ◽

10.1182/blood-2020-136548 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-36

Author(s):

Geffen Kleinstern ◽

J. Brice Weinberg ◽

Sameer A. Parikh ◽

Esteban Braggio ◽

Dennis P. Robinson ◽

...

Keyword(s):

Logistic Regression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Polygenic Risk Score ◽

Advisory Committees ◽

Polygenic Risk ◽

C Statistic

Background MBL is a precursor to chronic lymphocytic leukemia (CLL) and is subclassified into low-count (LC) MBL (absolute B-cell count<0.5x109/L) and high-count (HC) MBL (absolute B-cell count between 0.5 and 5x109/L). We previously reported that a polygenic risk score (PRS) based on a weighted average of 41 CLL-susceptibility variants was associated with risk of both MBL and CLL among a cohort of individuals from CLL families. Here we evaluate this PRS in an independent cohort of MBL and CLL individuals of European ancestry (EA), all of whom were ascertained agnostic to CLL family-history status. We also evaluate the PRS by MBL subtype (LC/HC), and in African American (AA) CLL cases and controls. Methods We genotyped 535 EA MBLs (139 HC-MBL, 396 LC-MBLs), 735 CLLs (640 EA, 95 AA), and 2,866 controls (2,631 EA, 235 AA) from the Mayo Clinic CLL Resource, Duke University, and Weill Cornell Medical College. We computed the CLL-PRS for each individual and used logistic regression to estimate odds ratios (OR) and 95% confidence intervals, adjusting for age and sex. To assess discriminatory accuracy, we computed the c-statistic. Among EA individuals, we calculated a trend test among LC-MBL, HC-MBL, and CLL risk using the P-value for heterogeneity from a polytomous logistic regression analysis. Moreover, we plotted a boxplot for the PRS among controls, LC-MBL, HC-MBL, and EA CLL, as well as for AA CLL cases and controls, and tested the statistical difference using the Kruskal Wallis test and Mann-Whitney test, respectively. Results We found a significant association of PRS with overall MBL risk (OR=1.87, P=1.1x10-28) with good discrimination (c-statistic=0.72). Significant associations were also found for LC-MBL (OR=1.75, P=7.5x10-19, c-statistic=0.72), HC-MBL (OR=2.22, P=1.4x10-17, c-statistic=0.74), and CLL of EA (OR=2.60, P=1.2x10-62, c-statistic=0.78), with a significant difference among these cohorts (Figure 1.A) and a significant positive trend across these cohorts (Pheterogeneity=8.4x10-6). Although we observed a 33% increased risk of CLL in AA (c-statistic=0.57), the PRS was borderline significant (P=0.07, Figure 1.B). Conclusion The CLL-PRS is a strong prediction-tool for risk of CLL and MBL among individuals of EA. Future studies are needed to improve the PRS for AAs including performing GWAS of AA in order to identify CLL-susceptibility SNPs that are more representative within known CLL loci and to discover novel CLL loci that are unique for AAs. Disclosures Parikh: GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Brander:Genentech: Consultancy, Honoraria, Other, Research Funding; Juno/Celgene/BMS: Other, Research Funding; MEI Pharma: Other, Research Funding; Ascentage: Other, Research Funding; ArQule: Consultancy, Other, Research Funding; NCCN: Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; AstraZeneca: Consultancy, Honoraria, Other, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other, Research Funding; Pfizer: Consultancy, Other; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Novartis: Consultancy, Other; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Tolero: Research Funding; Teva: Consultancy, Honoraria; DTRM: Other, Research Funding; BeiGene: Other, Research Funding; Novartis: Consultancy, Other; NCCN: Other; Verastem: Consultancy, Honoraria, Other, Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees. Furman:Acerta: Consultancy; AstraZeneca: Consultancy, Research Funding; Beigene: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Loxo Oncology: Consultancy; Oncotarget: Consultancy. Shanafelt:Mayo Clinic: Patents & Royalties: and other intellectual property; Genentech, Pharmacyclics LLC, an AbbVie Company, AbbVie, GlaxoSmithKline, and Merck: Research Funding.

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Single-Cell Multi-Omics Reveals Distinct Paths to Survival of Admixed BTKC481 Mutant Vs. Wild-Type Cells in Clinically Progressing Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-138374 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-42

Author(s):

Andrew Lipsky ◽

Danny Luan ◽

Shirley Chen ◽

Ronan Chaligne ◽

Neville Dusaj ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Single Cell ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Publicly Traded ◽

Cell Identity ◽

Single Cell Sequencing ◽

Bcr Signaling

Mutations in the kinase binding domain of BTK at position C481 are associated with resistance to BTK inhibitor (BTKi) therapy in chronic lymphocytic leukemia (CLL). Nearly half of patients manifesting clinical progression with these alterations exhibit a subclonal burden of resistance. Intriguingly, measured BTKC481 variant allelic fractions (VAF) are commonly lower than 10% [Ahn et al, Blood 2017]. This raises the important question of how BTKC481-mutated (MUT) and wildtype (WT) cells differ in their response to therapeutic challenge, and how low-burden MUT subclones facilitate escape from therapy. While the admixture of MUT and WT cells within the same individual presents an opportunity to directly study the downstream effects of subclonal resistance mutations, these cells cannot be separated through sorting. To overcome this limitation, we utilized Genotyping of Transcriptomes (GoT)-a strategy to jointly capture genotyping of a locus of interest together with whole transcriptomes at the single cell level (Fig. 1A). Importantly, GoT eliminates patient-specific and technical confounders, enabling direct linkage of BTK genotypes to transcriptional phenotypes. We applied GoT to 64,099 CD19+ cells across a cohort of seven patients with clinically progressive CLL found to have low-burden BTKC481 subclones (Fig. 1B). Samples were obtained at the time of progression and were screened for mutations in PLCG2. We genotyped 33.3% of cells, consistent with 34.0% of cells expressing BTK in whole transcriptome data. Clustering of the gene expression profiles showed that MUT and WT clones did not segregate by genotype in most (6/7) patients (Fig. 1C), implying a large degree of transcriptional similarity between MUT and WT cells, and further highlighting the need for multi-omics single-cell sequencing to directly link genotype to phenotype in this context. We note that the single exception with distinct genotypic clustering (CLL06, Fig. 1D) was driven by co-occurring large chromosomal aberrations within the MUT cells, including del(8p), which is associated with BTKi resistance [Burger et al, Nat Comm 2016]. CLL cells are known to cycle between the peripheral blood and protective microenvironmental niches, which is a process modulated by BTKi. We applied CXCR4/CD5 expression as a read out to these migration patterns [Chen et al, Leukemia 2016], and observed that MUT cells were comparatively enriched in CXCR4lowCD5hi CLL, which is associated with recent emigration from the stromal niche and increased BCR signaling (Fig. 1E). Conversely, WT cells were the majority of CXCR4hiCD5low CLL, which is associated with a resting peripheral cellular state and BCR downregulation, suggesting WT quiescence with limited stromal support. To explore the phenotypic changes associated with BTK mutations, we first measured the activity of reported BTKi response expression signatures. WT cells showed higher mean response scores compared to MUT (Fig. 1F), demonstrating that the WT cells preserve BTKi responsiveness. To unbiasedly interrogate transcriptional differences, we applied a set of 336 predefined gene modules representing B-cell cellular functions and processes. Enriched modules in MUT cells implicated restoration of B-cell receptor (BCR) signaling and recovery of CLL cell identity (NF-kB, IRF4, CD40, Fig. 1G), a finding also supported by de novo differential gene expression analysis (Fig. 1H), and gene set enrichment of differentially expressed genes (Fig. 1I). In contrast, WT cells showed increases in modules related to quiescence, hypoxia, cellular stress (HIF-1α, XBP1) and terminal B-cell development (Blimp-1). Intriguingly, changes in gene targets associated with restoration of NOTCH1 and IL4 activity were also observed. As these pathways are impaired in patients responding to BTKi and implicated in resistance [Del Papa et al, CCR 2019; Chen et al, ASH 2019], their re-emergence may reflect a cytoprotective strategy. In summary, we utilized multi-omics single-cell sequencing to identify the distinct transcriptional programs of admixed MUT and WT cells in subclonal BTKC481 progression. MUT cells showed robust escape from BTKi inhibition via increased immune receptor signaling and restoration of CLL cell identity program, while WT cells demonstrated a signature of hypoxia and stress response, with NOTCH1 and IL4 activation implicated as mechanisms of clonal persistence. Disclosures Trisal: Celgene: Current Employment, Current equity holder in publicly-traded company. Gandhi:Celgene: Current Employment, Current equity holder in publicly-traded company. Wiestner:Pharmacyclics LLC, an AbbVie Company, Acerta, Merck, Nurix, Verastem, and Genmab: Research Funding; NIH: Patents & Royalties: NIH. Allan:Acerta, Genentech, Abbvie, Sunesis, Ascentage, Pharmacyclics, Janssen, AstraZeneca, BeiGene: Consultancy; Celgene, Genentech, Janssen, TG Therapeutics: Research Funding; Abbvie, Janssen, AstraZeneca, Pharmacyclics: Honoraria. Furman:Genentech: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy, Research Funding; Acerta: Consultancy; Verastem: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy, Research Funding; Incyte: Consultancy; Janssen: Consultancy, Speakers Bureau; Oncotarget: Consultancy; Loxo Oncology: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

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