Akhtar, Sharoon

Abstract Background Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells in the peripheral blood, bone marrow, lymph nodes, and spleen. This is due to a combined effect of deferred apoptosis with slow, but persistent proliferation of malignant cells. In CLL, tumor-sustaining homeostasis is critically maintained by the ubiquitin proteasome system. The proteasome mediates degradation of various transcription factors such as TP53 as well as upholding a balance between the anti and pro apoptotic proteins of the BCL2 family. Previous studies have demonstrated that the clinical course of the disease is negatively associated with malfunctioning apoptotic pathways that result in increased levels of BCL2. Thus, identification and correction of defects that affect programmed cell death offer therapeutic vantage to reset and engage cell death pathways in CLL. Aim Examination of the anti-CLL properties of the investigational agent MLN2238 (Millennium Pharmaceuticals, Inc., Cambridge, MA) and its ability to inhibit the proteasomal machinery; induce CLL cell death and downregulate BCL2. MLN2238 activity was also investigated in conjunction with anti-CLL therapies such as fludarabine and dexamethasone along with the BH3 mimetic BCL2 inhibitor, AT-101 (Ascenta Pharmaceuticals, Malvern, PA). Methods CLL cells with >90% CD19+ tumor population were obtained from 28 patients with a confirmed diagnosis of CLL. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. Results MLN2238 inhibited the chymotrypsin-like proteasomal activity by more than 90% (p<0.005) in all patient samples without altering PSMB5 protein levels. Moderate to minimal inhibitory effect on caspase-like and trypsin-like proteasomal activities, respectively, was also noted. CLL cells showed a concentration dependent decrease in viability in response to treatment with MLN2238 at an IC50 of 50 nM. MLN2238 treated cells underwent apoptosis in a dose dependent manner with a median dose effect (cell death) observed in 42% of cells at 25 nM (range 10% - 54%) and 60% of cells at a 50 nM concentration (range, 25% - 73%). PARP-1 and caspase-3 cleavage along with an increase in MOMP was also noted after CLL cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. BCL2 downregulation was dose-dependent and was observed as early as 12 hours. We sought to determine whether directly disrupting BCL2 function with AT-101 could enhance the anti-CLL effects of MLN2238. When used at sub-IC50 concentrations, AT1-10 synergized with MLN2238 to induce CLL cell death. Synergy was also observed when MLN2238 was paired with the cytotoxic agent fludarabine, whereas the combination of MLN2238 and dexamethasone resulted in additive anti-CLL activity. Conclusion While PI have made an important impact in various B cell cancers, their role in CLL has not been well established. We investigated preclinically, a novel PI and noted that targeting the proteasome with MLN2238 resulted in lethal events in CLL cells, which were further enhanced by disruption of the BCL2 prosurvival pathway. Moreover, proteasome disruption sensitized CLL cells to the cytotoxic effect of fludarabine, an important therapeutic in CLL. These data provides the mechanistic basis for evaluation of MLN2238 in CLL through rationale design of drug combination strategies based on CLL biology. We would like to acknowledge the Leukemia and Lymphoma Society (A.C.-K. is a Leukemia and Lymphoma Scholar in Clinical Research) for their ongoing support. We are also grateful to Mary Ella Mahoney Davidson (Millennium Pharmaceuticals) for providing logistical support. Disclosures: Foran: Celgene: Research Funding.

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL. Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study. Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for >1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state. Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. Disclosures No relevant conflicts of interest to declare.

Abstract Background:The pathological behavior of Waldenströms macroglobulinemia (WM) cells relies on aberrant B-cell receptor (BCR), and Toll-like receptor (TLR) signaling and is intricately supported by malfunctions in initiation of apoptosis. In this dynamic, a complex shift between functionally active pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family occurs and bestows the malignant cells with a significant survival advantage. This defective apoptotic signaling is associated with aggressive clinical behavior, chemoresistance, and a poor prognosis in many B-cell cancers, including WM. Thus, BCR/TLR induced proliferation and abnormal apoptotic signaling due to dysregulation of Bcl-2 signaling, act in concert for the assurance of the malignant WM cells survival. Clinically, mitigation of BCR signaling with the first in class BTK inhibitor, ibrutinib, has shown promising results in WM and other B-cell cancers. However, clinical responses to the BTK inhibitor alone are less than optimal and development of ibrutinib-resistant disease is being recognized as an important and impending dilemma. We have previously shown the utility of disrupting pro-apoptotic Bcl-2 protein interactions in WM, chronic lymphocytic leukemia and multiple myeloma and that this functional interference results in re-sensitization (or heightened sensitivity) of tumor cells towards chemoimmunotherapy. As such, we examined the effects of ABT-199, a Bcl-2 specific small molecule inhibitor, in preclinical models of WM. Importantly; we conducted this investigation in proprietarily developed WM cells that have acquired resistance mechanisms towards either ibrutinib (IR WM subclones) or bortezomib (BR WM subclones). Materials:WM cell lines (BCWM.1, MWCL.1 and RPCI-WM1; WT cells) including their bortezomib resistant (BR) or ibrutinib resistant (IR) subclones were used in this study. Results: An initial comparison of Bcl-2 family members, Bcl-2, Mcl-1, BCL-xL, A1 (anti-apoptotic members), Noxa, Puma, Bim (pro-apoptotic initiators) and Bak and Bax (pro-apoptotic effectors), revealed that BR WM cells express increased levels of pro-apoptotic Bcl-2 family members, particularly Bcl-2, relative to WT cells. Similarly, A1 and Bcl-2 levels were up regulated in IR cell lines suggesting that the Bcl-2 mediated survival pathway is involved in resistance to both bortezomib and ibrutinib. Cell growth (MTS) assays using ABT-199 indicated that both BR and IR cells were equally sensitivity to Bcl-2 inhibition, with an IC50 ranging between 2 – 4mM. ABT-199 induced a dose dependent increase in MOMP with concomitant induction of apoptotic cell death, the latter confirmed by observation of PARP-1 cleavage. ABT-199 induced an intrinsic apoptotic program with the activation of caspase 9 and caspase 3 in all the cells tested. This data indicated the Bcl-2 pathway as being relevant and essential in WM cell lines even under drug resistant conditions. To evaluate the priming effect of ABT-199 on bortezomib and ibrutinib induced cell death, MTS assays were performed with these drugs in presence of IC25 concentrations of ABT-199. The addition of ABT-199 significantly reduced the IC50of both bortezomib and ibrutinib in WT, BR and IR cells. This suggested that re-sensitization of BR and IR cells towards either bortezomib or ibrutinib, respectively, was possible in presence of ABT-199. Further, combined addition of ABT-199 with bortezomib or ibrutinib showed synergistic cell death (combination index range: 0.004 – 0.6, CalcuSyn, Biosoft UK) in BR or IR WM cells, respectively. Conclusion: Dysregulation of Bcl-2 signaling is associated with the pathogenesis and promotion of drug resistance in B-cell cancers, including WM. Our report is first to highlight in WM preclinically, the antiproliferative effect of disrupting Bcl-2, through use of ABT-199. Altogether, our data suggests that therapeutic interference of Bcl-2 function with ABT-199 complements the anti-WM activity of ibrutinib or bortezomib in a synergistic manner and can in fact restore sensitivity of either drug in WM cells that are resistant to proteasome or BTK inhibitors. Focused mRNA and protein profiling analyses are currently underway to delineate the mechanisms accounting for synergy observed between concomitant Bcl-2/BTK inhibition as well Bcl-2/proteasome inhibition. Disclosures Ansell: Biothera: Research Funding. Martin:Janssen: Honoraria. Coleman:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Speakers Bureau.

AbstractPurpose:CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied.Experimental Design:We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT.Results:In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model.Conclusions:Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

SummaryCD38 is expressed on Waldenström macroglobulinaemia (WM) cells, but its role as a therapeutic target remains undefined. With recent approval of the anti‐CD38 monoclonal antibody, daratumumab (Dara), we hypothesized that blocking CD38 would be lethal to WM cells.In vitroDara treatment of WM cells (including ibrutinib‐resistant lines) elicited antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytotoxicity (CDC), antibody‐dependent cell phagocytosis (ADCP) and direct apoptosis.In vivo, Dara treatment was well tolerated and delayed tumour growth in RPCI‐WM1‐xenografted mice. CD38 is reported to augment B‐cell receptor (BCR) signalling; we noted that Dara significantly attenuated phosphorylated SYK, LYN, BTK, PLCγ2, ERK1/2, AKT, mTOR, and S6 levels, and this effect was augmented by cotreatment with ibrutinib. Indeed, WM cells, including ibrutinib‐resistant WM cell lines treated with the ibrutinib + Dara combination, showed significantly more cell death through ADCC, CDC, ADCP and apoptosis relative to single‐agent Dara or ibrutinib. In summary, we are the first to report thein vitroandin vivoanti‐WM activity of Dara. Furthermore, we show a close connection between BCR and CD38 signalling, which can be co‐targeted with ibrutinib + Dara to induce marked WM cell death, irrespective of acquired resistance to ibrutinib.

AbstractIn a continuing search for curcuminoid (CUR) compounds with antitumor activity, a novel series of heterocyclic CUR–BF2 adducts and CUR compounds based on indole, benzothiophene, and benzofuran along with their aryl pyrazoles were synthesized. Computational docking studies were performed to compare binding efficiency to target proteins involved in specific cancers, namely HER2, proteasome, VEGFR, BRAF, and Bcl‐2, versus known inhibitor drugs. The majority presented very good binding affinities, similar to, and even more favorable than those of known inhibitors. The indole‐based CUR–BF2 and CUR compounds and their bis‐thiocyanato derivatives exhibited high anti‐proliferative and apoptotic activity by in vitro bioassays against a panel of 60 cancer cell lines, more specifically against multiple myeloma (MM) cell lines (KMS11, MM1.S, and RPMI‐8226) with significantly lower IC50 values versus healthy PBMC cells; they also exhibited higher anti‐proliferative activity in human colorectal cancer cells (HCT116, HT29, DLD‐1, RKO, SW837, and Caco2) than the parent curcumin, while showing notably lower cytotoxicity in normal colon cells (CCD112CoN and CCD841CoN).

SummaryDeubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome‐associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b‐AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient‐derived WM tumour cells. Importantly, b‐AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib. In silico modelling identified protein residues that were critical for the binding of b‐AP15 with USP14 or UCHL5 and proteasome enzyme activity assays confirmed that b‐AP15 does not affect the proteolytic capabilities of the 20S proteasome β‐subunits. In vitro toxicity from b‐AP15 appeared to result from a build‐up of ubiquitinated proteins and activation of the endoplasmic reticulum stress response in WM cells, an effect that also disrupted the mitochondria. Focused transcriptome profiling of b‐AP15‐treated WM cells revealed modulation of several genes regulating cell stress and NF‐κB signalling, the latter whose protein translocation and downstream target activation was reduced by b‐AP15 in vitro. This is the first report to define the effects and underlying mechanisms associated with inhibition of USP14 and UCHL5 DUB activity in WM tumour cells.