Viralkumar Patel

Impact of bone marrow stromal cells on Bcl-2 family members in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world. High levels of Bcl-2 family anti-apoptotic proteins are responsible for apoptosis resistance. Besides anti-apoptotic proteins, the microenvironment provides substantial survival signals to CLL leukemic cells. However, in-depth knowledge on the role of individual Bcl-2 family members in the context of the microenvironment is still limited. We performed a comprehensive analysis of transcripts and proteins of 18 Bcl-2 family members using an “apoptosis array microfluidic card” in primary cells before and after stromal co-cultures. Our data showed that five of six anti-apoptotic members (excluding Bcl-b), two of three pro-apoptotic members (excluding Bok) and six of nine BH3-only members were present at detectable mRNA levels in CLL cells. Importantly, stromal-mediated extended survival of CLL cells was strongly associated with elevated global transcription. Upon co-culturing with stromal cells, there was an early response of an increase in anti- (2/5) and pro-apoptotic protein (3/8) transcripts on day 1, while an increase in anti-apoptotic proteins was observed on day 3, with no significant change in pro-apoptotic proteins. Our study revealed a differential pattern of expression of both transcripts and proteins following stromal co-cultures, proposing a significance of Bcl-2 family members in the stromal microenvironment.

Comparison of Acalabrutinib, A Selective Bruton Tyrosine Kinase Inhibitor, with Ibrutinib in Chronic Lymphocytic Leukemia Cells

Purpose: Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects. Experimental Design: Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine the effects on other kinases. Results: Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase-3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on the phosphorylation of BTK and downstream S6 and ERK kinases. In contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes. Conclusions: Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T cells. Clin Cancer Res; 23(14); 3734–43. ©2016 AACR.

Expression of executioner procaspases and their activation by a procaspase-activating compound in chronic lymphocytic leukemia cells.

Intrinsic and extrinsic apoptotic pathways converge to activate common downstream executioner caspases (caspase-3, -6, and -7), resulting in cell death. In chronic lymphocytic leukemia (CLL), neoplastic B cells evade apoptosis owing to the overexpression of survival proteins. We hypothesized that direct activation of procaspases could bypass the apoptosis resistance induced by the upstream prosurvival proteins. The procaspase-activating compounds (PAC-1), including B-PAC-1 (L14R8), convert inactive executioner procaspases to their active cleaved forms by chelation of labile zinc ions. Both at transcript and protein levels, primary CLL cells express high levels of latent procaspases (3, -7, and -9). B-PAC-1 treatment induced CLL lymphocyte death which was higher than that in normal peripheral blood mononuclear cells or B cells, and was independent of prognostic markers and microenvironmental factors. Mechanistically, B-PAC-1 treatment activated executioner procaspases and not other Zn-dependent enzymes. Exogenous zinc completely, and pancaspase inhibitors partially, reversed B-PAC-1-induced apoptosis, elucidating the zinc-mediated mechanism of action. The cell demise relied on the presence of caspase-3/7 but not caspase-8 or Bax/Bak proteins. B-PAC-1 in combination with an inhibitor of apoptosis protein antagonist (Smac066) synergistically induced apoptosis in CLL samples. Our investigations demonstrated that direct activation of executioner procaspases via B-PAC-1 treatment bypasses apoptosis resistance and is a novel approach for CLL therapeutics.

Preclinical combination of TP-0903, an AXL inhibitor and B-PAC-1, a procaspase-activating compound with ibrutinib in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in which apoptotic-resistant mature lymphocytes relentlessly accumulate in bone-marrow, lymph nodes and peripheral blood. In these body compartments, CLL B cells get survival signals mainly through the B-cell antigen receptor (BCR) pathway. The critical role of BCR signaling in CLL pathogenesis, i.e. proliferation, survival, adhesion, migration, and differentiation, is very well established. Bruton’s tyrosine kinase (BTK) is a key component of the BCR pathway and is required for CLL maintenance and development. BCR pathway inhibition through irreversible inhibition of BTK at Cys481 residue in its ATP-binding domain by ibrutinib has significantly improved the treatment outcome in CLL via disrupting the interactions with microenvironment. Phase 1 clinical trial of ibrutinib (PCI-32765) in relapsed or refractory CLL malignancy patients resulted in 70% overall response rate [1]. Phase 1b and 2 ibrutinib clinical testing in patients with relapsed and refractory disease also showed similar (70%) response rate which lead to breakthrough status for ibrutinib [2]. Outstanding outcome during clinical trials without untoward toxicity resulted in FDA approval of ibrutinib for patients with CLL who have received at least one prior therapy. Long-term follow-up suggested a revolutionary role of ibrutinib for CLL patients. Single-agent ibrutinib induced durable responses in the majority of CLL patients; however, these were mostly partial remissions, less than 10% achieved complete remission and, importantly, patients are not cured. A major part of the reason for partial remission is lymphocytosis after ibrutinib treatment which was due to the egress of CLL lymphocytes from lymph node niches. A key feature of ibrutinib therapy was early and dramatic reduction in lymph nodes with a parallel increase in CLL cells in blood circulation [2]. Ibrutinibmediated BTK inhibition does not induce cell death directly but impairs BCR-associated integrin-mediated adhesion and migration, which leads to CLL cells efflux from lymphoid tissues (lymph node microenvironment) to peripheral blood. In peripheral blood due to lack of sustained survival factors which are in lymphoid tissues, these lymphocytosed CLL cells slowly undergo cell death. However, this slow process might allow the opportunity of selection or outgrowth of resistant clones of CLL cells. In fact, during therapy, in many individuals, peripheral blood lymphocytosis was prolonged. Furthermore, some of the patients on continuous therapy with ibrutinib developed resistance to ibrutinib therapy [3]. Among these patients, mutation at the ibrutinib-binding site on BTK, mutation in additional molecules in the BTK axis such as PLCg2, and non BCR pathway modulations were identified [3]. Moreover driver mutation SF3B1 which is associated with poor prognosis also contributed to ibrutinib-acquired resistance [4]. Finally, increased Bcl-2 protein with a decline in Mcl-1 and Bcl-XL has also been observed and suggested as a survival mechanism for ibrutinib-treated CLL cells [5]. These data underscore a need to identify pharmacological agents that can synergistically partner with ibrutinib. Continuous and prolonged use of ibrutinib incurs a high cost of therapy. Finding optimal drug combinations would not only help achieve deeper responses but may also taper the high cost of chronic ibrutinib intake. TP-0903 is a selective inhibitor of Axl receptor tyrosine kinase. Axl receptor, a member of TAM (Tyro3, Axl and Mertk) family of receptor tyrosine kinases, is overexpressed in CLL and plays an oncogenic survival role [6]. Previously Sinha and colleagues [7] demonstrated

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkins B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

Apoptosis Pathways in Chronic Lymphocytic Leukemia: Role of the Microenvironment and Therapeutic Strategies

Chronic lymphocytic leukemia (CLL) is a B‐cell malignancy marked by defective apoptosis and apoptotic resistance. CLL lymphocytes accumulate in bone marrow, lymph nodes, and peripheral blood and receive survival signals through a diverse microenvironment in these body compartments. CLL microenvironment and cell interactions have been studied extensively. The microenvironment aggravates the antiapoptotic components in CLL cells and depletes the proapoptotic signaling that is essential to defending against apoptosis resistance. Ample research has been conducted to understand the involvement of apoptosis pathway proteins and the role of the microenvironment in the biology of leukemic cells. Major protein groups of the apoptosis pathway are the B‐cell lymphoma 2 (Bcl-2) family, inhibitor of apoptosis protein (IAP) family, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF-kB) signaling axis, tumor necrosis factor receptor superfamily (TNFRSF), B-cell receptor (BCR), death effector domain-containing proteins (DED) family, caspase activation and recruitment domain-containing proteins (CARD) family, and the caspase family. Here, we review the role of apoptosis pathway protein groups in the CLL microenvironment and strategies to counter survival signals of these groups as approaches in CLL therapy.

Abstract 2657: Elevated level of BCL-2 is the primary target for inhibition during duvelisib (IPI-145) therapy: ABT-199 neutralizes the resistance mechanism in chronic lymphocytic leukemia

Inhibition of BTK by ibrutinib, PI3K-delta with idelalisib, or PI3K-delta and gamma with duvelisib (IPI-145), all components of B-cell receptor (BCR) pathway, has significantly improved the treatment outcome in chronic lymphocytic leukemia (CLL) via disrupting the interactions with microenvironment. Though BCR network inhibitors induce durable remission in the majority of CLL patients, a proportion of patients that initially respond to treatment develop resistance or some show maintained lymphocytosis in the blood. Identifying and overcoming resistance mechanisms will be crucial for the most effective combinatorial use of these agents. Duvelisib is an orally bioavailable, highly potent small molecule inhibitor of p110δ and p110γ with KD values of 0.023 nM and 0.24 nM, respectively. Preclinical investigations with duvelisib overcame signals from PI3K/AKT/S6 pathway and promoted apoptosis in primary CLL cells (Balakrishnan et al, ASH 2013). Duvelisib is currently in a phase III trial in CLL. During first phase I study, we performed molecular investigations. PBMCs collected from CLL patients from Phase I study of duvelisib treatment (Pre/Day 0 and Post/Day 28) were subjected to RPPA analysis (n = 7). Interestingly, of the 141 proteins analyzed, Bcl-2 was maximally and significantly elevated in Day 28 samples (mean fold + SEM: 1.7 + 0.2; p = 0.015), suggesting that this could be in part the mechanism involved in resistance during therapy. Further analysis of protein expression by immunoblotting confirmed that Bcl-2 protein was elevated in post-treatment samples (1.3 + 0.1; p = 0.086; n = 7). This was in conjunction with elevated levels of Bcl-2 transcripts analyzed by mRNA array (TaqMan Human Apoptosis - 93 genes) assay (3.0 + 0.4; p = 0.002) and RT-PCR (1.9 + 0.2; p = 0.003), while other anti-apoptotic genes (Bfl-1, Mcl-1, Bcl-w Bcl-g, Bcl-b, Bcl-xL) were unchanged. Importantly, ex-vivo incubations of pre- and post- duvelisib samples with 3 nM ABT-199, a highly selective clinically promising Bcl-2 protein antagonist, induced significantly greater apoptosis in post-therapy samples (79%) in comparison to pre-treatment (58%) samples suggesting that Bcl-2 is the primary target for inhibition during duvelisib intake (n = 5; p = 0.041). In addition, duvelisib induced sensitivity on ex-vivo post- duvelisib clinical samples (n = 15) was specifically towards ABT-199 and produced significantly more apoptosis in the presence of ABT-199 (45%, p Citation Format: Viralkumar M. Patel, Kumudha Balakrishnan, Renato Guerrieri, William Wierda, Susan O9Brien, Varsha Gandhi. Elevated level of BCL-2 is the primary target for inhibition during duvelisib (IPI-145) therapy: ABT-199 neutralizes the resistance mechanism in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2015-2657

Abstract 3338: Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chronic lymphocytic leukemia (CLL) is a B-cell malignancy and leukemia cells accumulate in bone marrow, lymph node, and peripheral blood due to decreased apoptosis. High levels of Bcl-2 family anti-apoptotic and inhibitors of apoptosis proteins (IAPs) in leukemic lymphocytes are primarily responsible for defective apoptosis in CLL. Both intrinsic caspase-9-mediated and extrinsic caspase-8-dependent apoptosis pathways activate downstream procaspase-3 to active casapce-3. In normal (n=4) as well as CLL lymphocytes (n=9), there is consistent expression of procaspase-3. However, active caspase-3 was present in normal lymphocytes but not in CLL cells suggesting that CLL cells have defective endogenous apoptosis. Hence, we hypothesized that compounds that can directly activate the functional ability of procaspase-3, should overcome the checkpoints of cell death and trigger apoptosis in CLL lymphocytes. Procaspase activating compounds (PAC; Vanquish Oncology) convert executioner procaspases to their active forms by chelating labile zinc on these enzymes. We have tested L14R8, a potent analogue of PAC, in primary CLL cells. The IC50 of L14R8-induced apoptosis in CLL was 7.7μM (n=12, 24hrs). L14R8 (10 μM) induced apoptosis as early as 6 hrs along with mitochondrial outer membrane permeabilization (MOMP). The drug significantly induced apoptosis in CLL patient samples (n=4) cultured in suspension cultures (60%, p=0.023,) as well as CLL lymphocytes under microenvironmental protection of marrow stromal cells (40%, p=0.011) albeit at a lower rate. Mechanistically, L14R8-treatment reduced uncleaved procaspases-3 and -9 expression and increased cleaved active caspases-3 and -9 expression (n=3). Consistent with the mechanism of action of L14R18 on terminal caspases, WT as well as cells lacking BAX and BAK (mouse embryo fibroblasts) were equally sensitive to this agent. Consistent with L14R8 mechanism, addition of exogenous Zinc ion completely reversed L14R8-mediated apoptosis of CLL lymphocytes (p=0.0063, n=4), but did not affect staurosporin-induced apoptosis in these lymphocytes. Moreover, L14R8-induced 86% (range 58-90%, n=4) cell death in CLL lymphocytes, which was reduced by pan caspase-inhibitors, Z-VAD to 58% (range 32-79%) and Q-VD-OPh to 35% (range 20-55%) cell death, which indicated the mechanistic recovery of L14R8-mediated apoptosis. Smac (the inhibitor of IAPs) protein expression was increased in CLL cells after treatment with L14R8. With respect to the therapeutic index, at 24 hrs, L14R8 induced 79% apoptosis in CLL cells (range 46%-97%, n=14) and only 18% (16-28%, n=3) in lymphocytes from healthy donors. We conclude that L14R8 induced apoptosis through activation of procaspase-3 to active caspase-3 in CLL cells. L14R8 and other PAC provide therapeutic index and may be developed into targeted therapeutics for patients with CLL. *Funded by Vanquish Oncology Citation Format: Viralkumar M. Patel, Kumudha Balakrishnan, William Wierda, Varsha Gandhi. Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2013-3338

Abstract 1490: Impact of bone marrow microenvironment on mRNA expression of genes in the apoptotic pathway in CLL cells

Chronic lymphocytic leukemia (CLL) is thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports revealed that CLL cells accumulate due to both an intrinsic defect in apoptosis and extrinsic signals delivered by the microenvironment. Although several entities of bone marrow stroma cell (MSC) have been unveiled, the critical components involved in CLL survival remain obscure. In the present study, we used low density microarray analysis to identify the essential apoptotic genes (n=93) that are involved in cell survival. Primary cells obtained from CLL patients (n=12) were co-cultured with MSC (Nktert, human stromal cell line that mimics bone marrow microenvironment) in a ratio of 100:1. At the end of the incubation (12hr, d1, and d3), the expression profile of 93 genes as well as the percent apoptosis were evaluated. When CLL lymphocytes were co-cultured on MSC for 6 days, the viability of CLL cells, measured by Annexin/PI staining, increased by 6-27% at d1-d6 (n=9, p 3 H]uridine incorporation (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1490. doi:1538-7445.AM2012-1490

Abstract 516: Marrow stromal cell rendered stability to chronic lymphocytic leukemia cells is partly mediated by Pim kinase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chronic lymphocytic leukemia (CLL) was thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports reveal that at least 1% of CLL cells undergo replication in marrow and the accumulation is not merely due to intrinsic defect in apoptosis but in part due to extrinsic signals delivered by microenvironment. Although several entities of marrow stromal cell (MSC) microenvironment have been elucidated, the critical elements involved in CLL survival remain obscure. Pim proteins are constitutively active Ser/Thr kinases and to date Pim-1, -2, -3 have been identified. Their activity on target proteins influences a number of cellular processes such as transcription, translation, proliferation and survival. Previously, we elucidated that Pim kinases are highly expressed in CLL cells compared to normal lymphocytes [Blood 114:4150, 2009]. Based on this background, we hypothesized that Pim and their substrates may essentially contribute to MSC-driven CLL cell survival. To test this, we co-cultured CLL lymphocytes with NKTert stromal cells (a human MSC line) that mimic marrow microenvironment. Evaluation of Pim in CLL cells by immunoblotting (n=10) revealed that Pim-1 and -2 expression increased with supporting MSC, with more pronounced increase in Pim-2. A similar trend was observed in total and phospho cMyc (Ser62, n=8) suggesting that Pim is a co-activator of MYC and MYC is a Pim-driven transcription factor, and their levels largely correlate. This is consistent with the observed increase in the global RNA synthesis (uridine incorporation) in CLL cells co-cultured with MSC. Histone H3, a transcriptional activator and a Pim target, is important for MYC driven transcription. In CLL cells co-cultured with NKTert cells, phospho Histone H3 (Ser10) diminished significantly starting as early as 1 hr (n=10). The mechanism underlying the decrease and changes at the early time points are under investigation. In conjunction with transcription, translation is also governed by Pim kinases through phosphorylation of translation suppressor protein 4E-BP1. In this regard, our preliminary data shows that in CLL cells 4E-BP1 phosphorylation status was not affected by MSC. This is in concordance with the steady-state levels of total protein synthesis (leucine incorporation) in presence of stromal support. CLL cells remained replicationally quiescent on MSC (Ki67 staining) and hence cell cycle substrates of Pim (p21, p27, cdc25a and c) were not analyzed. In terms of survival (annexin/PI binding), the viability of CLL cells on MSC increased by 10-25% at day 4, 5, 6 of incubation (n=4, 6, 5, respectively). Consistent with this data, in CLL cells Bad phosphorylation (Ser112), a Pim substrate, was elevated with stromal support. Taken together, we demonstrate that MSC-rendered stability to CLL cells by Pim kinases is in part mediated by increased transcription and disrupted apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 516. doi:10.1158/1538-7445.AM2011-516