BCR Pathway Research Articles

Microenvironment regulates the expression of miR-21 and tumor suppressor genes PTEN, PIAS3 and PDCD4 through ZAP-70 in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.

Ironclad: A randomized phase III study of ibrutinib (Ibr) or no consolidation following autologous hematopoietic stem cell transplantation (AutoHCT) for relapsed/refractory activated-B-cell (ABC) subtype diffuse large B-cell lymphoma (DLBCL).

TPS7566 Background: Relapsed DLBCL in the rituximab era portends a poor prognosis with only about 25% of patients achieving long-term disease control following 2ndline therapy and AutoHCT. Patients with the ABC subtype have an inferior prognosis at diagnosis than those with GC and are overrepresented at relapse. In order to improve outcomes in ABC-DLBCL, we designed a study targeting disease pathobiology at the time of AutoHCT. Ibr has a safety profile allowing combination with cytotoxic chemotherapy and has single-agent activity with a 37% response rate in patients with relapsed/refractory ABC-DLBCL. Methods: This is an intergroup, randomized, placebo-controlled phase III study combining Ibr or placebo with high-dose chemotherapy during conditioning with AutoHCT and for 12 months following AutoHCT. Pts with relapsed or refractory DLBCL have tissue submitted centrally for real-time review and subtype assignment by GEP (Frederick Laboratory). Key eligibility criteria include no more than 3 prior regimens, no active CNS involvement, no need for long-term anticoagulation, and no progression on prior Ibr. Pts with chemosensitive ABC-DLBCL are randomized to Ibr 560 mg or placebo with BEAM or CBV chemotherapy until day 0. After engraftment, pts receive Ibr 560 mg daily or placebo for 12 additional cycles. Pts with progressive disease on placebo can cross over to Ibr monotherapy. An initial safety cohort of 6 pts is being enrolled to evaluate the tolerability of Ibr with concurrent BEAM and CBV therapy. The primary endpoint is superior 2-year PFS (Ha/H0: 67% vs 50%). Secondary endpoints include time to count recovery, post-transplant response rates, OS, PFS, and incidence of 2arymalignancies. In correlative studies, we will assess the prognostic and predictive role of pre-transplant FDG-PET in the setting of Ibr or placebo therapy, the role of emergent BCR pathway mutations, double hit genetics, and pharmacogenetic determinants on treatment outcome and toxicities. We expect to accrue 296 patients over 4 years. An Alliance/BMT-CTN study: NCT02443077 Clinical trial information: NCT02443077.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells invitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both invivo and invitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

Inhibiting pathways involved in B‐cell development enhances sensitivity of B‐cell acute lymphoblastic leukemia to glucocorticoids

Glucocorticoids, such as dexamethasone (dex), are used in combination chemotherapy for leukemias and lymphomas. Dex activates the glucocorticoid receptor (GR), which in turn regulates genes that induce cell death. Although dex is a remarkably effective therapeutic, some patients are insensitive to it, and others develop resistance. From among the thousands of GR‐regulated genes, it is not known which are required to induce cell death, nor how resistance arises in most cases. To understand which GR regulated genes drive lymphoid cell death, and which signaling pathways modulate dex‐sensitivity, we took an integrated functional genomic approach. We first identified sets of dex‐regulated genes by measuring differential gene expression in a panel of childhood B‐cell acute lymphoblastic leukemia (B‐ALL) primary samples and cell lines. We then performed a next‐generation genome‐wide shRNA screen to identify genes that affect growth and sensitivity to dex in B‐ALL. By comparing regulation and effect on sensitivity we essentially validated all genes en masse in B‐ALL, and were able to show that only 10% of dex‐regulated genes contributed to cell death. These genes predominantly function in transcription regulation and B‐cell development. This screen also identified cellular pathways that modulate dex‐induced death, including the B‐cell receptor pathway (BCR), a key checkpoint in B‐cell development. By integrating the gene expression and shRNA data sets we identified a double‐negative feedback loop between GR and PI3Kδ, a lynchpin in the BCR and IL7R pathways. Inhibition of PI3Kδ attenuated phosphorylation of GR, enhanced dex‐induced gene regulation, and restored sensitivity to even highly resistant B‐ALL specimens. Thus, this work elaborates with high resolution fundamental mechanistic details of GR function, and, in doing so, identifies numerous opportunities for enhanced lymphoid‐specific combination chemotherapy.Support or Funding InformationNIH, NSF, Carver Charitable Trust, V Foundation

Molecular Subtyping in Diffuse Large B Cell Lymphoma: Closer to an Approach of Precision Therapy.

It has become clear that there is immense biological heterogeneity in diffuse large B cell lymphoma (DLBCL). Developing technology has allowed better characterization of patient subsets at a molecular level, allowing for a link of phenotype and clinical outcomes to oncogenic mechanisms and biologic signatures. Cell of origin and double hit status are able to identify aggressive subsets, with molecular profiling allowing for a clearer understanding of biologic pathways that contribute to cellular resistance to conventional treatment in these subsets. Although the standard treatment for DLBCL remains R-CHOP or R-CHOP-like therapy at present, rational drug targets have been established with novel classes of drugs under investigation. In germinal center (GC) DLBCL, mechanisms of therapeutic interest include anti-apoptosis mediated by BCL-2, PI3K/AKT/mTOR, and EZH2, whereas drug interventions are directed at BCR, NF-κB, and/or JAK-STAT pathways in activated B cell (ABC) DLBCL. There is also evidence for cooperation of various oncogenic pathways in these subsets. As such, we are arguably on the verge of shifting to a more tailored approach using single and combinatorial strategies-this, however, relies on prioritizing the exploration of biomarkers for patient selection for validating novel treatment strategies.

Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment.

Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTKC481S-transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NF?B. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

Dual SYK/JAK Inhibition Overcomes Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Ibrutinib (a BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Further, cerdulatinib blocked proliferation of ibrutinib-sensitive and ibrutinib-resistant primary CLL cells and of BTKC481S-transfected cells. These anti-tumor effects are correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition. DisclosuresCoffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

The Bruton's Tyrosine Kinase (BTK) Inhibitor ARQ 531 Effectively Inhibits Wild Type and C481S Mutant BTK and Is Superior to Ibrutinib in a Mouse Model of Chronic Lymphocytic Leukemia

Introduction: The Bruton's tyrosine kinase inhibitor ibrutinib improves survival in chronic lymphocytic leukemia (CLL) compared to standard chemotherapy or immune therapy. In a subset of patients, somatic mutation (C481S) of its binding site results in acquired resistance to ibrutinib therapy with poor clinical outcome. ARQ 531 is an ATP competitive, orally bioavailable, potent inhibitor of BTK and other relevant kinases including the majority of Src and Tec family kinases. Herein we present preclinical data with ARQ 531 in CLL including C481S mutated BTK and its efficacy versus ibrutinib in the TCL1 mouse model of CLL.Methods: Potency of ARQ 531 and its binding kinetics were measured in enzymatic and Surface Plasmon Resonance (SPR) binding assays. Primary CLL B cells were negatively selected and treated with 1μM ARQ 531. BCR signaling was investigated by immunoblot following a 1 hour drug incubation. CLL cells migrating towards CXCL12 and CXCL13 after 4 hours across a 5.0 micron transwell insert were counted by flow cytometry. Annexin V and propidium iodide flow cytometry was used to measure CLL viability over a range of drug concentrations and time. CpG mediated CLL cell activation was measured by CD40 and CD86 expression by flow cytometry. In vivo investigation utilized B6 mice engrafted with 1E7 CD5+/CD19+ TCL1 lymphocytes via tail vein injection. Mice were randomized to treatment with vehicle, ARQ 531, or ibrutinib following the establishment of a CD5+/CD19+ population >10% in peripheral blood.Results: At 72 hours the viability of CLL cells treated with 0.1µM, 1.0µM, or 10.0µM ARQ 531 was found to be 94%, 67%, and 50% that of untreated samples, respectively (p=0.76, p<0.001, p=0.016) in comparison to the 71% relative viability seen with 1µM ibrutinib (p<0.001). ARQ 531 demonstrates dose dependent inhibition of BTK, AKT, and ERK phosphorylation. Stimulation through TLR9 by CpG ligand increases expression of the activation markers CD40 and CD86; ARQ 531 decreased CpG induced expression of CD40 by 25% and CD86 by 40% (p=0.022, p=0.041). ARQ 531 in the presence of CXCL12 or CXCL13 decreased migration by 51% and 66%, respectively (p=0.015, p=0.025), below baseline migration of untreated CLL.Unlike ibrutinib, ARQ 531 inhibits both wild type and C481S BTK with an IC50 less than 1nM in a biochemical assay, and inhibits phosphorylation of C481S mutated BTK in LV125 transfected HEK293T cells. Cytotoxicity was also observed in CLL cells isolated from patients with C481S BTK mutations, where the viability of cells treated with 1µM ARQ 531 was 72% that of untreated cells at 72 hours (p=0.038). SPR binding assay showed a residence time of 51 minutes in wild type BTK and 56 minutes in C481S mutated BTK.The TCL1 mouse model displays active BCR signaling and responds to treatment with the BTK inhibitors ibrutinib and acalabrutinib (Ponader S, 2012; Herman SEM, 2015). Following establishment of leukemia in the TCL1 transfer model, we randomized mice to treatment with vehicle (n=14), 25 mg/kg ibrutinib (n=6), 50 mg/kg ARQ 531 (n=14) or 75 mg/kg ARQ 531 (n=14) given by daily gavage. ARQ 531 significantly improved survival over both ibrutinib and vehicle (median survival: 36 days vehicle, 53 days ibrutinib, not reached by 74 days 50mg/kg and 75 mg/kg ARQ 531, p<0.0001). Mice receiving ARQ 531 have consistently lower lymphocyte counts on microscopic examination and reduced CD5+/CD19+ populations via flow cytometry compared to vehicle or ibrutinib. Additionally, we have observed increases in absolute neutrophil count over time in mice receiving ARQ 531. Pharmacokinetic studies are pending; however, in a single oral dose study of 10mg/kg in monkeys, the bioavailability was 72.4% with a CMax of 9µM and a half-life greater than 24 hours.Conclusion: The BTK inhibitor ARQ 531 is a potent inhibitor of BTK with promising activity both in vitro and in vivo. Multi-targeted inhibition of cytokine, chemokine, and BCR pathways by ARQ 531 decreases activation, migration, and viability of CLL cells. Unlike ibrutinib, ARQ 531 inhibits activation of C481S mutated BTK variants and maintains cytotoxicity in ibrutinib resistant clones. Additionally, ARQ 531 has demonstrated remarkable efficacy in an in vivo TCL1 adoptive transfer model, improving survival to a greater extent than ibrutinib and potentially restoring granulocyte production. These data justify continued preclinical work and development to facilitate transition to clinical trials. DisclosuresEathiraj:ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

Lymphoid-like Environment, Which Promotes Proliferation and Induces Resistance to BH3-Mimetics, Is Counteracted By Obinutuzumab in MCL: Biological Rationale for the Oasis Clinical Trial

Mantle cell lymphoma (MCL) accumulates in lymphoid organs but disseminates early on in extranodal tissues. However, there is relatively little information regarding the nature of surrounding cells and soluble factors and the resulting molecular regulations induced in MCL. Further investigations that integrate the key role of the microenvironment are now needed to overcome tumor drug resistance in protective niches. The benefits of such an approach have been recently reinforced by studies reporting that selective inhibition of BTK or PI3Kδ, critical kinases of the BCR and CXCR4 pathways, prevents the homing of MCL, leading to peripheral lymphocytosis. However, the consequences of cell egress in the peripheral blood on proliferation and survival are still unclear and need further investigations.In spite of the significant level of the proliferation index Ki67 in secondary lymphoid organs and the elevated percentage of circulating malignant cells, we did not detect any proliferating peripheral blood MCL cells, which were arrested in the G1-phase of the cell cycle. The proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem. In situ, lymphoma B-cells are in close contact with cells of mesenchymal or immune origin. To determine whether stromal (hMSC) or lymphoid-like (CD40L) interactions could support survival and proliferation of MCL, primary circulating MCL cells from 21 patients were cocultured several weeks ex vivo. In all samples tested we showed that CD40L signal, but not hMSC, induced cell-cycle progression, which was amplified by a MCL-specific cytokine cocktail, defined by assessment of cytokine receptor expression in situ. Using this system, we were able to maintain proliferation of primary MCL cells for several weeks, and most MCL samples remained dependent on the extrinsic signal over time.Of major interest, using RNA-seq, we showed that 70% of genes induced ex vivo are expressed in lymph nodes, confirming the relevance of the coculture. We further observed that our model recapitulated in situ MCL molecular signatures such as proliferation, BCR/NFkB and survival signatures.Among the genes related to survival, coculture resulted in anti- and pro-apoptotic Bcl-2 family member modulation. We demonstrated that an anti-apoptotic Bcl-xL protein increase was associated with a striking downregulation of pro-apoptotic Bim, Bax and Bak. Of note, this unbalanced regulation seemed to be specific to MCL cells as it did not occur in naïve CD5+ B cells or memory B cells. We provided evidences that Bcl-xL upregulation was responsible for loss of mitochondrial priming and drug resistance. Using BH3-profiling, we showed a dynamic sequestration of the BH3-only activator Bim by Bcl-xL proteins at the mitochondrial level. In addition, we showed that the mitochondrial priming directly correlated with the sensitivity toward BH3-mimetics (venetoclax) and alkylating drugs (bendamustine). Our results thus highlight molecular basis for microenvironment-dependent modulation of the mitochondrial priming and consequent drug resistance in primary MCL cells.Thus, whereas venetoclax is highly efficient in inducing apoptosis in peripheral blood MCL cells, tumor cells protected into their niches could be resistant and in fine induce relapse. Using our ex vivo model, we further demonstrated that, in rational combinations, anti-CD20 antibody (obinutuzumab) can suppress survival and overcomes drug resistance to venetoclax exploiting NFkB/Bcl-xL-specific dependence of primary MCL cells. Moreover, ibrutinib, which mediates indirect Bcl-xL down-modulation upon BTK-dependent lymphocytosis, could also increase in vivo venetoclax efficacy. Our ongoing OAsIs Trial for MCL patients (obinutuzumab, ibrutinib and venetoclax, NTC#02558816) will rapidly determine in vivoefficacy.In summary, we reported here the development of a reproducible ex vivo coculture model for primary MCL cells. This model has provided new insights into microenvironment-dependent proliferation and Bcl-2 family regulation, which are central components of survival and drug resistance. Our increased understanding of intrinsic abnormalities, the development of highly selective inhibitors and integration of the microenvironment offer new opportunities to design mechanism-based strategies that should overcome drug resistance in MCL and potentially other B cell malignancies DisclosuresMoreau:Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria.

Exploring the Functional Relevance of BTK Beyond Chronic Lymphocytic Leukemia (CLL) Cells: BTK Expression in Non-Malignant Immune Cells of the Microenvironment Mediates CLL Development and Progression In Vivo

Background:Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression.Methods:Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines.Results:To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1.Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. DisclosuresWoyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

The Landscape of Dynamic Genetic Changes in Ibrutinib-Treated CLL

Cancer evolution poses a significant challenge to the ability of therapy to eliminate malignant cells. In chronic lymphocytic leukemia (CLL), clonal evolution was reported as a key feature of disease relapse after standard chemoimmunotherapy and after targeted therapy with the BTK inhibitor ibrutinib. To study potential selection of pre-existing sub-clones, we analyzed changes in clonal architecture at early time points in patients receiving ibrutinib therapy. We therefore performed in-depth longitudinal analysis of CLL samples collected from 59 patients treated with ibrutinib alone (n=44) or in combination with rituximab (n=15).Across this cohort, we performed whole-exome sequencing (WES) on a median of 3 samples per patient with median coverage of 105x (range 50-225x). Pretreatment samples were available for all patients, with post-treatment samples at 1 and 6 months for more than 85% of patients. Additional samples were available at 2, 3, or 12 months for 22 patients. The median age of the cohort was 65 (range 33-85) years. Thirty-one patients (53%) had del(17p) by FISH, 37 patients (63%) had IGHV unmutated CLL and 32 patients (54%) had relapsed or refractory disease. We observed a median of 56 (range 17-201) somatic silent and non-silent single nucleotide variants (SNVs) and insertions and deletions (Indels) per patient. The number of SNVs and indels did not differ between patients receiving ibrutinib alone or with rituximab (Wilcoxon, P=0.2). Thirteen patients had somatic mutations of BCR pathway genes prior to treatment, including one with CARD11-L251P, previously reported to confer resistance to ibrutinib in diffuse large B-cell lymphoma.Comparing pre-treatment and the latest samples within the first year (range 56-357 days, median 168), 26 of 59 (44%) CLLs showed significant changes in cancer cell fraction (CCF) over time, defined as a shift > 5% CCF of the clone with the greatest change (BH-FDR <0.1). These included 11 CLLs with a clone that significantly increased in size, 9 with a clone that significantly decreased in size, and 6 with significant reciprocal rises and falls of different clones consistent with a branched evolutionary pattern. The median CCF change during the observation period was 24% (range 7-89%). Thus, even within a short period of treatment exposure, dense temporal sampling of CLLs reveals that ibrutinib therapy commonly leads to clonal shifts.The likelihood for clonal shifts was not impacted by whether patients were treated with ibrutinib alone or with rituximab (45% vs. 40%, P=0.77), nor by exposure to prior therapy (47% vs. 41% with and without prior therapy, P=0.79). We observed trends towards higher likelihood of early clonal evolution in CLLs with unmutated IGHV status, with TP53 somatic aberrations, and those with a pre-treatment subclonal driver (subclones containing CHD2, BIRC3, SF3B1, MAP2K1, CARD11, XPO1, TP53, PTPN11, KRAS, HIST1H1E, ATM). Finally, patients with a decrease in ALC at 6 months compared to pre-treatment were more likely to exhibit significant CCF shifts (P=0.01). This last observation suggests that effective treatment activity and resulting therapeutic bottlenecks indeed contribute to the shift in clonal proportion within the first year of ibrutinib therapy.Relapse samples were available for 10 CLL cases, 6 of whom showed early clonal shifts during the first year of therapy. These included 2 cases of transformation to prolymphocytic leukemia (PLL), 2 with Richter transformation (RT) and 6 cases of progressive CLL disease. All relapse cases were clonally related to the pre-treatment samples. Mutations in BTK or PLCg2 were found in 5 cases by either WES or deep targeted sequencing of known resistance hotspots (both PLL, one RT, two progressive CLL). The remaining 5 cases showed marked clonal shifts suggestive of selection of other resistant genotypes.Altogether, targeted therapy with ibrutinib is associated with overall clonal stability in more than half of CLLs over the first year of treatment. Nevertheless, in many patients, clonal selection occurs even during the first months of therapy and may provide clues regarding the future evolutionary and clinical outcome of the disease. DisclosuresFarooqui:Merck: Employment. Burger:Pharmacyclics, LLC, an AbbVie Company: Research Funding; Gilead: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Roche: Other: Travel, Accommodations, Expenses; Portola: Consultancy. Wiestner:Acerta Pharma: Research Funding; Pharmacyclics: Research Funding. Wu:Neon Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Rituximab Preferentially Eliminates BCR Signaling Proficient Chronic Lymphocytic Leukemia B Cells In Vivo

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab.We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P<0.0001) compared to CLL cells from the same patient circulating in the blood stream for a longer time (CXCR4brightCD5dim), and we showed that CD20 is up-regulated by stromal cell interactions through the binding of SDF-1α to CXCR4 (P<0.001). We further hypothesized that higher levels of CD20 on CXCR4dimCD5bright cells will make them the primary target for rituximab in vivo. To investigate this, we obtained peripheral blood samples from CLL patients (N=17) before rituximab administration (day 0) and 24 hours (day 1) after rituximab administration (375 mg/m2, single agent). This showed that rituximab primarily and nearly completely eliminates the CXCR4dimCD5bright subpopulation (8.3 % pre-rituximab vs. 2.1 % post-rituximab, P<0.0001), which harbors the highest levels of CD20 (P<0.005).It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P<0.005). This was also coupled with higher responsiveness of CXCR4dimCD5bright cells to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Moreover, CXCR4dimCD5bright cells also had higher levels of CD19 (1.8-fold, P<0.0001), which is an important docking molecule in PI3K signaling, and its higher levels lower the threshold for BCR signaling activation.Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib.This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz DisclosuresNo relevant conflicts of interest to declare.

Prospect & progress of venetoclax in treating chronic lymphocytic leukemia

ABSTRACTIntroduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the overexpression of BCL2, a pro-survival protein responsible for impaired apoptosis, tumor initiation, disease progression, and drug resistance. Venetoclax is a potent, second generation, orally bioavailable, BCL2 inhibitor.Areas covered: In early phase clinical trials, venetoclax has demonstrated remarkable activity in heavily pre-treated patients and those with poor prognostic features. Furthermore, it has shown efficacy and safety when combined with immunotherapy and chemotherapy, which has prompted phase III investigation. The most common adverse events are related to myelosuppression, infection, and diarrhea. Tumor lysis syndrome is the most concerning adverse event associated with venetoclax, but can be mitigated with risk-adapted prophylaxis.Expert opinion: The treatment landscape for CLL is evolving from chemoimmunotherapy to involve targeted biologic therapies, based on a greater understanding of the pathogenesis of this disease. Venetoclax provides an effective alternative to BCR pathway antagonists, with a distinct mechanism of action and adverse event profile.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Abstract Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death &amp; ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (&amp;gt;1.5-fold) reveals the involvement of the BCR (PLCG2, BTK &amp; GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 &amp; RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1&amp; PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 &amp; 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

Abstract 346: Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies

Abstract Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.

Association of activated BCR and PI3K pathways with responses and longer PFS to single-agent copanlisib in heavily pretreated patients with indolent and aggressive NHL.

e19063Background: Copanlisib, a novel pan-class I PI3K inhibitor with predominant activity against α and δ isoforms, has shown promising single agent anti-tumor activity in a phase 2 study in heavi...

Venetoclax activity in CLL patients who have relapsed after or are refractory to ibrutinib or idelalisib.

7519Background: Patients (pts) with chronic lymphocytic leukemia (CLL) who relapse after or become refractory (R/R) to BCR pathway inhibitors have poor outcomes. Venetoclax (VEN) is a selective, or...

An autoimmune risk variant in PTPN22 alters tolerance at multiple checkpoints during naive B cell development

Abstract A missense risk variant (R620W) in the tyrosine phosphatase PTPN22 is strongly associated with multiple autoimmune diseases. To model this variant in vivo, we generated knock-in mice expressing the analogous variant (R619W) in murine Ptpn22. Expression of the variant significantly altered lymphocyte function and led to the development of systemic autoimmunity. Further, B lineage-restricted variant expression was sufficient to promote autoimmunity (Dai et al. JCI 2013). In the current study, we performed a detailed assessment of the impact of the variant on B cell tolerance, including its impact on the establishment of the naive repertoire. Utilizing a bone marrow (BM) chimera model, variant expressing B cells exhibited a competitive advantage over wild-type (WT) cells at multiple checkpoints, beginning in the BM and continuing in the periphery. Interestingly, while follicular (FM) B cells maintain this competitive advantage, marginal zone (MZ) B cells do not. In addition, using single cell BCR cloning, we observed a similar differential phenotype, where FM B cells are highly enriched for self-reactivity, while MZ B cells are less autoreactive than WT controls. Using a range of analyses to assess cell signaling, surface receptor expression, cell cycle and gene expression, we provide evidence that the risk variant regulates a complex interplay between the BCR and CD40 co-receptor pathways, culminating in a uniquely autoreactive pre-GC repertoire with important implications for subsequent development of autoimmunity.

Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia.

Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.

Molecular modeling studies on series of Btk inhibitors using docking, structure-based 3D-QSAR and molecular dynamics simulation: a combined approach.

Bruton tyrosine kinase (Btk) is a non-receptor tyrosine kinase. It is a crucial component in BCR pathway and expressed only in hematopoietic cells except T cells and Natural killer cells. BTK is a promising target because of its involvement in signaling pathways and B cell diseases such as autoimmune disorders and lymphoma. In this work, a combined molecular modeling study of molecular docking, 3D-QSAR and molecular dynamic (MD) simulation were performed on a series of 2,5-diaminopyrimidine compounds as inhibitors targeting Btk kinase to understand the interaction and key residues involved in the inhibition. A structure based CoMFA (q (2) = 0.675, NOC = 5, r (2) = 0.961) and COMSIA (q (2) = 0.704, NOC = 6, r (2) = 0.962) models were developed from the conformation obtained by docking. The developed models were subjected to various validation techniques such as leave-five-out, external test set, bootstrapping, progressive sampling and rm (2) metrics and found to have a good predictive ability in both internal and external validation. Our docking results showed the important residues that interacts in the active site residues in inhibition of Btk kinase. Furthermore, molecular dynamics simulation was employed to study the stability of the docked conformation and to investigate the binding interactions in detail. The MD simulation analyses identified several important hydrogen bonds with Btk, including the gatekeeper residue Thr474 and Met477 at the hinge region. Hydrogen bond with active site residues Leu408 and Arg525 were also recognized. A good correlation between the MD results, docking studies and the contour map analysis are observed. This indicates that the developed models are reliable. Our results from this study can provide insights in the designing and development of more potent Btk kinase inhibitors.