Wnt5a Induces ROR1-Dependent Upregulation of MMP9 and Enhanced Invasiveness in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells recirculate between blood and lymphoid tissue compartments, where they receive growth/survival signals from accessory cells within the lymphoid-tissue microenvironment. Such trafficking requires leukemia cells to pass through endothelial barriers and to transverse the extracellular matrix (EM), a process that is facilitated by matrix metallopeptidases (MMP). We co-cultured CLL cells with marrow mesenchymal stromal cells (MSCs) at a physiologic oxygen tension (e.g., 5% O2 in N2). Under such conditions we found that MSCs could enhance leukemia-cell expression of MMP9, a 92 kDa type IV collagenase and key MMP involved in EM degradation. Unexpectedly, however, we found circulating CLL cells with high-level expression of the onco-embryonic protein, ROR1 (designated ROR1Pos), also had high-level expression of MMP9 despite not having immediate contact with such accessory cells. Moreover, we found that circulating ROR1Pos CLL (N = 12) had significantly greater levels of MMP9 than blood CLL cells with low-to-negligible levels of ROR1 (ROR1Neg CLL, N = 10) (P < 0.001). Short-term culture of ROR1Pos CLL in serum-free media significantly reduced their expressed levels of MMP9, unless we added exogenous Wnt5a, a non-canonical Wnt factor and ligand for ROR1 that we found expressed at significantly higher levels in the plasma of patients with CLL than in age-matched adults (Yu et al., J Clin Invest, 2015). Treatment of ROR1Pos CLL cells with Wnt5a enhanced their expression and release of MMP9 and their capacity to invade Matrigel in a Boyden-Chamber Assay; such effects could not be inhibited by inhibitors of B-cell receptor/chemokine signaling (e.g. Ibrutinib), but could be blocked by cirmtuzumab, a humanized mAb specific for ROR1 that can inhibit leukemia-cell ROR1-signaling in patients with CLL (Choi et al., Cell Stem Cell, 2018). Silencing expression of MMP9 with siRNA or treatment with a MMP9-specific inhibitor (CAS 1177749-58-4) could inhibit the capacity of Wnt5a to enhance the invasive capability of CLL cells, indicating that MMP9 plays a major role in facilitating CLL-cell invasiveness. Targeted siRNA-mediated silencing of cytoskeletal proteins HS1/cortactin, which complex with ROR1 in response to Wnt5a (Hasan et al., Leukemia, 2018; Hasan et al., Leukemia, 2017), also impaired the capacity of Wnt5a to enhance expression and release of MMP9 in ROR1Pos CLL. Collectively, these studies indicate that Wnt5a can induce ROR1-signaling leading to enhanced expression of MMP9, which can facilitate invasion of the EM. Moreover, inhibitors of ROR1 signaling, such as cirmtuzumab, can repress Wnt5a-induced CLL-cell expression of MMP9, potentially impairing the homing of ROR1Pos CLL to their protective niches within the lymphoid microenvironment. Disclosures Kipps: AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson & Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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High-Level ROR1 and BCL2, Cancer-Stemness, and BCL2 Mutations Associate with Venetoclax Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-131426 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 476-476

Author(s):

Emanuela M. Ghia ◽

Laura Z. Rassenti ◽

Michael Y. Choi ◽

Elvin Chu ◽

George F. Widhopf II ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Gene Expression Signature ◽

Lymphocytic Leukemia ◽

Complex Karyotype ◽

Cancer Stemness ◽

Advisory Committees ◽

Group B ◽

High Level ◽

Cell Stem Cell

Venetoclax (Ven) is an inhibitor of BCL2 that is highly active in patients (pts) with chronic lymphocytic leukemia (CLL), effecting remissions without detectable minimal residual disease (MRD), particularly when used in combination with an anti-CD20 mAb (Seymour et al., N Engl J Med, 2018). However, pts can have persistent detectable MRD (i.e. ≥10-4 CLL cells by flow cytometry) after ≥1 year (yr) of Ven therapy (V-Rx); such pts are at risk for developing progressive disease (PD) even with continued V-Rx (Kater et al., J Clin Oncol, 2019). Evaluation of CLL cells from such pts may define biologic markers for pts who are likely to have persistent MRD after 1 yr of V-Rx and elucidate potential mechanism(s) of Ven resistance. We examined the CLL cells of pts (N=13) who had persistent MRD after ≥1 yr of V-Rx; 8 developed PD after a median time of 2 yrs on V-Rx and were designated as being in subgroup A1. Pts who had persistent MRD without PD after ≥1 yr of V-Rx were designated as being in subgroup A2 (N=5). For comparison we examined the pre-treatment (pre-Rx) CLL cells of pts who cleared MRD within 1 yr of therapy (N=5), and designated such pts as being in group B. The CLL cells of most pts expressed unmutated IGHV (i.e. 7/8 pts in A1, 3/5 pts in A2, and 5/5 pts in B). However, a high proportion of the pts with MRD after ≥1 yr of V-Rx had pre-Rx CLL cells with a complex karyotype and del17p (i.e. 5/8 pts in A1 and 2/5 pts in A2); whereas none of pre-Rx CLL cells of group B had a complex karyotype or del17p. We examined CLL cells for intracellular BCL2 and surface ROR1, which prior studies showed were correlative in CLL (Rassenti et al., Proc Natl Acad Sci U S A, 2017). The pre-Rx CLL cells of pts from subgroups A1 and A2 expressed significantly higher levels of ROR1 and BCL2 than the pre-Rx CLL cells of group B (P=0.03 and 0.0002, respectively, Mann-Whitney test). Furthermore, the CLL cells of pts with PD on V-Rx expressed significantly higher levels of ROR1 and BCL2 than the already high-levels expressed by the pre-Rx CLL cells of these same pts (P=0.002 and 0.01, respectively, Paired t test). We did not observe temporal changes in ROR1 or BCL2 in serial CLL samples collected over a comparable time interval from a comparator group of pts with adverse cytogenetics who did not receive V-Rx. We performed RNA sequencing with a mean of 70-million reads per sample on negatively-selected pre-Rx CLL cells from each pt, and on the isolated CLL cells from each of 6 pts in subgroup A1 when they had PD on V-Rx. Transcriptome analyses revealed the cancer-stemness gene-expression signature influenced by ROR1-signaling and associated with poorly-differentiated cancers (Choi et al., Cell Stem Cell, 2018; Malta et al., Cell, 2018) was significantly enriched in pre-Rx CLL of pts in subgroups A1 and/or A2 compared to group B (A1 and/or A2 vs. B had FDR q values of <0.001). We also found the transcriptomes of CLL cells from pts with PD on V-Rx had a significantly greater enrichment in the cancer-stemness gene-expression signature than that of the pre-Rx CLL cells of the same pts (FDR q value <0.001)! We identified the BCL2G101V mutation found earlier (Blombery et al., Cancer Discov, 2019) in the CLL cells of 3 of 6 pts with PD in subgroup A1 at allelic frequencies of less than 20%; this BCL2G101V mutation was not detected in pre-Rx CLL samples. We identified a new nonsynonymous BCL2 mutation at an allelic frequency of 49.3% in the CLL cells of 1 pt with PD who lacked the BCL2G101V mutation; this pt's pre-Rx CLL cells did not harbor detectable levels of this BCL2 mutation, which we deduce alters the BCL2 BH3-binding pocket. In summary, this study reveals that pts with CLL cells having complex cytogenetics, del17p, high-level expression of ROR1 and BCL2, and/or transcriptomes enriched for cancer-stemness may be at greater risk for having persistent MRD at ≥1 yr of V-Rx. Furthermore, the CLL cells of pts who develop PD on V-Rx have significantly higher levels of ROR1 and BCL2, BCL2 mutations, and transcriptomes with greater enrichment of the cancer-stemness signature than that of CLL cells from the same pts prior to V-Rx, implying that CLL cells resistant to Ven have greater cancer-cell de-differentiation. Because of the high frequency of mutations in BCL2 for pts with PD on V-Rx, strategies targeting ROR1 (Choi et al., Cell Stem Cell, 2018), rather than higher doses of Ven, may be more effective in mitigating the risk of PD in high-risk pts treated with Ven-based regimens. Disclosures Choi: Oncternal: Research Funding; Gilead: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Rigel: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding, Speakers Bureau. Kipps:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson & Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees.

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Activation of NF-Kappa B-p62-NRF2 Signaling Supports the Survival of CLL Cells That Express High Levels of ROR1

Blood ◽

10.1182/blood-2018-99-115564 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3122-3122

Author(s):

Elsa Sanchez-Lopez ◽

Emanuela M. Ghia ◽

Laura Antonucci ◽

Laura Z. Rassenti ◽

Yun Chen ◽

...

Keyword(s):

Mrna Expression ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Nuclear Factor ◽

High Level Expression ◽

Advisory Committees ◽

Resistance To Therapy ◽

High Level ◽

Inducible Genes

Abstract Recent studies have linked the autophagy adaptor p62/SQSTM1 to tumorigenesis via NRF2 signaling. Increased accumulation p62 is associated with disease progression in many cancers, however its expression in CLL has yet to be investigated. Importantly, p62, encoded by the SQSTM1 gene, mRNA expression and protein accumulation is regulated by nuclear factor-kappa B (NF-κB), a key transcription factor for CLL survival. In addition, CLL cells express receptor tyrosine kinase-like orphan receptor 1 (ROR1), an oncoembryonic protein that also is expressed on cancer cells of numerous malignancies, including those reported to show accumulation of p62. As in solid-tumor malignancies, high-level expression ROR1 in CLL is associated with enhanced disease-progression and shorter overall survival (Cui B, et al, Blood 25:2931, 2016). Furthermore, recent studies found that activation of Wnt5a-ROR1 cascade induces NF-κB activation and increased expression of NF-κB target genes in CLL (Chen, Y et al, submitted ASH Abstract, 2018). Additionally, CLL cells exhibit increased basal activation of nuclear factor erythroid 2-related factor 2 (NRF2), which supports antioxidant and detoxifying responses that enhance resistance to cytotoxic drugs. We hypothesized that high-level expression of ROR1 in CLL may influence the accumulation of p62, which in turn could lead to activation of NRF2-signaling to enhance CLL-cell survival and resistance to therapy. We found that CLL cells with high-level surface expression of ROR1 (ROR1HIGH) had significantly greater accumulation of p62 than ROR1LOW CLL cells (p=0.003). Elevated p62 accumulation was accompanied by enhanced protein and mRNA expression of NRF2 targets, including NQO1 (p=0.009), GPX2 (p=0.019), GSTM1 (p=0.001), SOD1 (p=0.001) and MDM2 (p=0.001) (n=6 ROR1LOW and n=13 ROR1HIGH). We analyzed published gene expression data (GSE13204, Kohlmann A, et al BJH 142:802, 2008), of 448 CLL cases. We designated CLL cells with expression levels of ROR1 above the medium (n=224) as ROR1HI and cases with ROR1 expression below the medium value as ROR1LO (n=224), We confirmed that SQSTM1 (p=0.01), NQO1 (p=0.0001) and HMOX1 (p=0.002) were significantly overexpressed in ROR1HI CLL patients. In addition, we segregated GSE13204 dataset into two groups: ROR1>90% (n=45), representing the 10% of patients who had CLL cells with highest levels of ROR1 mRNA, and ROR1<10% (n=45), representing the 10% of patients who had CLL cells with lowest levels of ROR1 mRNA. Consistently, SQSTM1 (p=0.009), NQO1 (p=0.02) and HMOX1 (p=0.008) were significantly overexpressed in ROR1>90%. We performed gene set enrichment analysis (GSEA) to investigate whether NRF2 inducible genes (Malhotra D, et al, Nucleic Acids Research 38(17):5718, 2010) were expressed at higher levels in ROR1HI or ROR1>90% compared with ROR1LO or ROR1<10% CLL. In both comparisons, the GSEA revealed that NRF2 inducible genes were enriched in CLL cells with high ROR1 mRNA (FDR q of 0.01 and 0.06, respectively). Collectively, these data indicate that p62-NRF2 cascade is upregulated in CLL cells with high expression of ROR1 and suggest that it may play a role in the enhanced proliferation observed in ROR1HIGH CLL cells. Similarly, ROR1HIGH CLL cells and MEC1-ROR1 CLL cell line that express high protein level of NQO1 were resistant to ABT-199 (p<0.05 and p<0.001 respectively). Such agent induces the accumulation of reactive oxygen species (ROS), which are detoxified by products of the NRF2-activated antioxidant response. The role of NQO1 in protection from cell death was confirmed using a specific pro-drug, designated as 29h, which becomes active after being metabolized by NQO1 (p<0.05). Furthermore, treatment with 29h overcame NQO1-mediated resistance to ABT-199 (venetoclax) (p<0.05), resulting in PARP cleavage and accumulation of cytoplasmic cytochrome C, markers of apoptosis. This study illustrates a previously unknown and intricate signaling network through which ROR1-NF-κB-p62-NRF2 cascade may enhance disease progression and resistance to therapy in CLL B cells that express high levels of ROR1. Disclosures Kipps: Genentech Inc: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; 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; Celgene: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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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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Impact of Idelalisib Treatment Interruption with or without Dose Reduction on Outcomes in Relapsed / Refractory Indolent Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-124541 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5468-5468

Author(s):

Shuo Ma ◽

Rebecca J Chan ◽

Lin Gu ◽

Guan Xing ◽

Nishan Rajakumaraswamy ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Dose Reduction ◽

Clinical Outcomes ◽

Research Funding ◽

Treatment Interruption ◽

Lymphocytic Leukemia ◽

Hodgkin's Lymphoma ◽

Advisory Committees

Introduction: Idelalisib (IDELA) is the first-in-class PI3Kδ inhibitor and is approved as a monotherapy for relapsed or refractory (R/R) follicular lymphoma and in combination with rituximab for R/R chronic lymphocytic leukemia (CLL). We previously evaluated IDELA treatment interruption as a mechanism to mitigate treatment-emergent adverse events (TEAEs) and found that limited interruption with clinically appropriate re-challenging resulted in superior clinical outcomes. These findings did not comprehensively address the potential confound of interruptions inherently being associated with longer duration of therapy (DoT). Furthermore, the compound effect of IDELA dose reduction together with treatment interruption on IDELA efficacy was not assessed. Objectives: 1) To evaluate whether the benefit of IDELA interruption is retained in patients on therapy >180 days, a duration previously found to be associated with longer overall survival among patients who discontinued IDELA due to an AE; and 2) To compare clinical outcomes of patients who reduced IDELA dosing in addition to interrupting IDELA with those of patients who interrupted IDELA without additional dose reduction. Methods: Using data from Gilead-sponsored trials of patients with R/R indolent non-Hodgkin's lymphoma (iNHL) treated with IDELA monotherapy (N=125, Gopal et al., N. Engl. J. Med., 2014) or with R/R CLL treated with IDELA + anti-CD20 (N=110, Furman et al., N. Engl. J. Med., 2014; and N=173, Jones et al., Lancet Haematol., 2017), DoT, progression-free survival (PFS), and overall survival (OS) were compared between patients on IDELA therapy >180 days with vs. without interruption and between patients who experienced Interruption and Dose Reduction (IDR) vs. patients who experienced Interruption but NoDose Reduction (INoDR) at any point during IDELA treatment. Interruption was defined as missing at least one IDELA treatment day due to an AE and dose reduction could have occurred before or after the first interruption. PFS and OS were estimated using the Kaplan-Meier method and were compared using a log-rank test. Results: Sixty-nine of 125 patients with R/R iNHL (55.2%) and 222 of 283 patients with R/R CLL (78.4%) remained on IDELA therapy >180 days with 29 (42.0%) and 103 (46.4%) of them, respectively, experiencing interruption on or after day 180 (Table 1). The proportions of patients with interruption before day 180 were similar within each of these populations. Among patients on therapy >180 days, those with treatment interruption on or after 180 days had a longer median (m) DOT than patients without interruption (Table 1). Both PFS and OS were longer in CLL patients who interrupted compared to those who did not interrupt (mPFS=28.9 mos. vs. 17.3 mos. and mOS=not reached [NR] vs. 40.4 mos. for with interruption vs. without interruption, respectively, Table 1 and Figure 1). In patients with iNHL, no difference was observed in PFS or OS between patients who interrupted vs. those who did not (Table 1). Of patients who experienced at least one AE-induced interruption at any point during IDELA therapy (n=63 iNHL and n=157 CLL), 47 iNHL patients (74.6%) and 84 CLL patients (53.5%) also had dose reduction. Two iNHL patients (1.6%) and 5 CLL patients (1.8%) had IDELA dose reduction but no interruption. Both iNHL and CLL patients with IDR experienced a similar PFS compared to patients with INoDR (mPFS=16.5 mos. vs. 14.2 mos. for iNHL and 21.8 mos. vs. 22.1 mos. for CLL with IDR vs. INoDR, respectively, Table 2). However, OS was longer in both iNHL and CLL patients with IDR compared to INoDR (mOS=61.2 mos. vs. 35.3 mos. for iNHL and NR vs. 42.4 mos. for CLL, respectively, Table 2; CLL patients shown in Figure 2). Discussion: IDELA treatment interruption is not associated with rapid clinical deterioration, as observed with some B-cell receptor signaling pathway inhibitors. No clear relationship between IDELA DoT and frequency of interruption was observed. When normalized for DoT >180 days, IDELA treatment interruption retained its clinical benefit in the CLL population. When utilized together with IDELA interruption, dose reduction did not lead to inferior clinical outcomes but instead extended OS in both iNHL and CLL populations. Adherence to treatment interruption and dose reduction guidance as outlined in the IDELA USPI may optimize IDELA tolerability and efficacy for patients with iNHL and CLL. Disclosures Ma: Janssen: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Research Funding; Abbvie: Research Funding; Juno: Research Funding; Incyte: Research Funding; Xeme: Research Funding; Beigene: Research Funding; Novartis: Research Funding; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Kite: Consultancy; Acerta: Research Funding; Bioverativ: Consultancy; Genentech: Consultancy. Chan:Gilead Sciences, Inc.: Employment, Equity Ownership. Gu:Gilead Sciences, Inc.: Employment. Xing:Gilead Sciences, Inc.: Employment. Rajakumaraswamy:Gilead Sciences, Inc.: Employment. Ruzicka:Gilead Sciences, Inc.: Employment. Wagner-Johnston:Gilead: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Jannsen: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

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BAX-Mutated Clonal Hematopoiesis in Patients on Long-Term Venetoclax for Relapsed/Refractory Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-137775 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Piers Blombery ◽

Ella R Thompson ◽

Xiangting Chen ◽

Tamia Nguyen ◽

Mary Ann Anderson ◽

...

Keyword(s):

Advisory Committee ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Clonal Hematopoiesis ◽

Eliza Hall Institute ◽

Hall Institute ◽

Over Time

Venetoclax (Ven) is an effective element of treatments for chronic lymphocytic leukemia (CLL) with high response rates observed in the upfront and relapsed/refractory (R/R) settings. In addition to inducing apoptosis in CLL cells, Ven also induces apoptosis within normal and malignant myeloid lineage populations (accounting for its efficacy in the treatment of acute myeloid leukemia). We investigated the effects of Ven outside the target tumor compartment in patients (pts) with CLL receiving long-term continuous Ven and make the novel observation of the development of BAX-mutated clonal hematopoiesis in this heavily pre-treated patient group. 92 pts with CLL receiving continuous non time-limited Ven have been treated at our institutions on clinical trials. Of these, 41 had sufficient (>6 mo) follow up (median 70; range 14-95 mo) and suitable samples available for further analysis. 38/41 (93%) pts had received previous treatment with alkylators and/or fludarabine. In order to assess the non-CLL compartment in these 41 pts we identified those with peripheral blood or bone marrow aspirate samples taken during deep response to Ven demonstrating either minimal (<5%) or no CLL involvement by flow cytometry (sensitivity 10-4). We initially performed unique molecular index (UMI)-based targeted next generation sequencing of apoptosis pathway genes as well a panel of 60 genes recurrently mutated in lymphoid and myeloid malignancy. From these 41 pts we identified mutations in the apoptosis effector BAX in samples from 12 (29%). 20 different BAX mutations were observed across these 12 pts at variant allele frequencies (VAF) consistent with their occurrence in the non-CLL compartment. Mutations included frameshift, nonsense, canonical splice site and missense mutations occurring in key structural elements of BAX consistent with a loss-of-function mechanism (Fig 1A). Interestingly, an enrichment of missense and truncating mutations predicted to escape nonsense mediated decay were observed at the C-terminus of the BAX protein affecting the critical α9 helix. Mutations in this region have previously been shown in cell lines to cause aberrant intracellular BAX localization and abrogation of normal BAX function in apoptosis (Fresquet Blood 2014; Kuwana J Biol Chem 2020). For comparison, NGS targeted sequencing for BAX mutations was performed on samples from cohorts of pts with (i) myeloid or lymphoid malignancy (n=80) or (ii) R/R CLL treated with BTK inhibitors (n=15) after a similar extent of preceding chemotherapy. Neither of these cohorts had previous exposure to Ven. BAX mutations were not detected in any samples from these pts. Longitudinal sampling from pts on Ven harboring BAX mutations in the non-CLL compartment was performed to further understand compartment dynamics over time (in 9 pts over 21-93 months of follow up). Multiple pts demonstrated a progressive increase in VAF of single BAX mutations over time to become clonally dominant within the non-CLL compartment and with observed VAFs consistent with their presence in the myeloid compartment. Mutations in other genes implicated in clonal hematopoiesis and myeloid malignancy including ASXL1, DNMT3A, TET2, U2AF1 and ZRSR2 were also detected in these pts samples. Targeted amplicon single cell sequencing (Mission Bio) demonstrated the co-occurrence of clonally progressive BAX mutations within the same clones as mutations in DNMT3A and ASXL1 as well as the existence of further BAX mutations at low VAF outside these dominant clones which remained non-progressive over time (Fig 1B). In addition, fluctuations in the presence and VAF of myeloid-disease associated mutations was noted with Ven exposure. In aggregate these data are consistent with the existence of a selective pressure within the myeloid compartment of these pts and an interplay of BAX with other mutations in determining survival and enrichment of these clones over time with ongoing Ven therapy. In summary, we have observed the development of BAX-mutated clonal hematopoiesis specifically in pts with CLL treated with long-term Ven. These data are consistent with a multi-lineage pharmacological effect of Ven leading to a survival advantage for clones harboring BAX mutations within the myeloid compartment during chronic Ven exposure. Finally, our data support the further investigation of BAX mutations as a potential resistance mechanism in myeloid malignancies treated with Ven. Disclosures Blombery: Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Seymour:Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Mei Pharma: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Nurix: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tam:Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; BeiGene: Honoraria. Huang:Servier: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Genentech: Research Funding. Wei:Janssen: Honoraria, Other; Walter and Eliza Hall Institute: Patents & Royalties; AMGEN: Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Other: Advisory committee; Pfizer: Honoraria, Other: Advisory committee; Macrogenics: Honoraria, Other: Advisory committee; Abbvie: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Genentech: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Speakers Bureau; Astra-Zeneca: Honoraria, Other: Advisory committee, Research Funding. Roberts:Janssen: Research Funding; Servier: Research Funding; AbbVie: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties.

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DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-123475 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2742-2742

Author(s):

Christian Hurtz ◽

Gerald Wertheim ◽

Rahul S. Bhansali ◽

Anne Lehman ◽

Grace Jeschke ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Negative Regulator ◽

Advisory Committees ◽

Normal Tissues ◽

Pharmacologic Inhibition

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

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The Broad Spectrum of TP53 Variants in CLL: NGS Analysis of 573 Pathogenic TP53 Variants

Blood ◽

10.1182/blood-2019-130907 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3021-3021

Author(s):

Gregory Lazarian ◽

Floriane Theves ◽

Myriam Hormi ◽

Virginie Eclache ◽

Stéphanie Poulain ◽

...

Keyword(s):

Tumor Progression ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Snp Analysis ◽

Advisory Committees ◽

Exon 2 ◽

Tp53 Mutations ◽

Mutational Status

TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency <10%. Even if they were predominantly found in polymutated cases, presence of only low VAF (<10%) mutations was evidenced in 74 (21%) patients (50 patients with a single TP53 mutation and 24 patients with more than one). All these cases would have been missed by conventional sequencing. Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients. Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more). Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations. Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment. Disclosures Leblond: Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.

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

Blood ◽

10.1182/blood-2020-137085 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-9

Author(s):

Shanye Yin ◽

Gregory Lazarian ◽

Elisa Ten Hacken ◽

Tomasz Sewastianik ◽

Satyen Gohil ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

Dna Binding ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Bcr Signaling ◽

Experimental Group

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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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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The Defective Proliferation of Vgamma9Vdelta2 T Cells to Zoledronic Acid In Chronic Lymphocytic Leukemia (CLL) Is a Powerful Time to First Treatment (TFT) Predictor Associated with the IGHV Mutational Status

Blood ◽

10.1182/blood.v116.21.3602.3602 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3602-3602

Author(s):

Marta Coscia ◽

Candida Vitale ◽

Silvia Peola ◽

Chiara Riganti ◽

Daniela Angelini ◽

...

Keyword(s):

T Cells ◽

Zoledronic Acid ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Advisory Committees ◽

Mutational Status ◽

Intermediate Metabolites ◽

Stimulation Of

Abstract Abstract 3602 The clinical course of chronic lymphocytic leukemia (CLL) depends on the intrinsic tumor cell features but also on the interactions between tumor cells, local microenvironment and host immunity. The interplay between CLL cells and conventional αβ T cells has already been investigated in details, whereas very little is known about the role of Vgamma9Vdelta2 T cells. These cells have intrinsic antitumor properties which can be further enhanced by stimulation with aminobisphosphonates such as zoledronic acid (ZA) via monocytes or other antigen-presenting cells. ZA targets the mevalonate (Mev) pathway and induces the intracellular accumulation and release of intermediate metabolites, like isopentenyl pyrophosphate (IPP), which are very similar to the natural ligands of Vgamma9Vdelta2 T cells. In this study we have performed a phenotypic and functional analysis of Vgamma9Vdelta2 T cells in 93 untreated CLL patients and correlated the results with intrinsic CLL cell features and clinical outcome. Stimulation of peripheral blood mononuclear cells with ZA induced Vgamma9Vdelta2 T cell proliferation in 47/93 patients (responders, R), but not in 46/93 patients (non-responders, NR). Vgamma9Vdelta2 T-cell subset distribution of R CLL was similar to healthy donors, whereas effector memory (EM) and terminally differentiated effector memory (TEMRA) cells predominated in NR CLL. A significant association was found between the R/NR status of Vgamma9Vdelta2 T cells and the mutational status of the tumor IGHV genes: 77% of R patients were M, whereas 70% of UM patients were NR. To test the hypothesis that this difference reflected a different activity of the Mev pathway in M and UM CLL cells, we performed a bioinformatic elaboration of data obtained from gene expression profiling of CLL cells and a biochemical quantification of the Mev pathway in CLL cells including the intermediate metabolites farnesyl pyrophosphate (FPP) and IPP, and the final product cholesterol. The Mev pathway was significantly more active in UM than in M CLL cells, suggesting that the IPP overproduction by UM CLL cells is responsible for a chronic stimulation of Vgamma9Vdelta2 T cells leading to their differentiation into EM and TEMRA cells. This biochemical-driven immunoediting process has clinical implications. After a median follow-up of 46 months from diagnosis, univariate analysis identified R status as a predictor of reduced TFT (NR: 59 months vs R: not reached; p=0.01). The R/NR status also allows to further identify two subsets in UM CLL patients (R-UM and NR-UM) with significantly different TFT (NR-UM: 32 months vs R-UM: not reached; p=0.02). In multivariate analysis, Binet stage (P=0.027), IGHV mutational status (p=0.016), R/NR status (p=0.007), high and low-risk cytogenetic abnormalities (p<0.001) and lymphocytosis at the time of diagnosis (p<0.001) were independent TFT predictors. In conclusion, we have identified a novel TFT predictor based on a putative interaction between the Mev pathway of CLL cells and Vgamma9Vdelta2 T cells. These results further strengthen the importance of tumor-host immune interactions in CLL evolution. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia:Novartis: Honoraria, Research Funding.

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