ALBERT

Description: Responses to single-agent ibrutinib (IBR) or venetoclax (VEN) in Chronic Lymphocytic Leukemia (CLL) or Mantle Cell Lymphoma (MCL) are often incomplete suggesting drug combinations are needed to overcome de novo and acquired resistance. We previously reported synergistic cytotoxicity for the IBR+VEN combination in CLL/MCL cells ex vivo and have initiated an IBR+VEN clinical trial in MCL (NCT02419560). However, ex vivo analysis of patient samples showed de novo resistance even to the IBR+VEN combination in some samples, consistent with recent clinical experience (Tam et al. N Engl J Med, 2018). We noted that sensitivity to IBR+VEN was lower in CLL cells showing an "activation" phenotype (CD5+/19+/69+) that can be induced by extracellular factors in vivo, suggesting that the microenvironment could induce drug tolerance in the cancer cells.This was supported by our finding that ex vivo exposure of CLL/MCL samples to a mixture of microenvironmental agonists (CpG-ODN, sCD40L, and IL10; "agonist mix") induced significant loss of sensitivity to IBR+VEN (Jayappa et al. Blood Adv., 2017). Here we show that various microenvironmental agonists, including of innate immunity, are able to reduce sensitivity to IBR+VEN as well as other pro-apoptotic drugs, generating a multi-drug tolerant phenotype. To explore drug tolerance, we performed flow cytometric analysis of apoptosis induced by IBR+VEN in CD5+/19+cancer cells in CLL/MCL PBMCs cultured with agonists of TLRs, NOD1/2, CD40, and IL10R and various stromal cells. Tolerance to IBR+VEN as determined by resistance to apoptosis was noted in most samples cultured with TLR9 agonist CpG-ODN, sCD40L, or Jurkat cells expressing CD40L. IL10 and HUVEC induced modest levels of drug tolerance in a few CLL/MCL samples, and TLR1/2, TLR7, and NOD1/2 agonists were effective only in MCL samples. Prior exposure to CpG-ODN enhanced the ability of sCD40L to induce proliferation and drug tolerance and vice versa in CLL/MCL, predicting mutually reinforcing interactions in vivo. We noted that CLL cells exposed to CpG-ODN displayed higher levels of CD40 and downstream signaling proteins (TRAF2, RelB, and p100/52). Conversely, CD40L induced a modest increase in NF-kB transcription factors, providing a possible mechanism for mutual reinforcement. Using flow cytometric analysis of apoptosis, we noted tolerance to several pro-apoptotic agents (bendamustine, fludarabine, and inhibitors of Mcl-1, Bcl-xL, Bcl-2, and Bcl-2/Bcl-xL) including IBR+VEN in CD5+/19+cells from CLL samples treated with agonist mix, showing development of multi-drug tolerance in these cells. CpG-ODN or agonist mix induced NFkB-dependent over-expression of pro-survival proteins (Mcl-1 and Bcl-xL), and increased ratio of these to cognate pro-apoptotic proteins. This increased expression of pro-survival proteins underlies the multi-drug tolerant phenotype. Consistently, multi-drug tolerance was rescued with inhibitors of NFkB (BMS345541 or Bortezomib) or drug combinations simultaneously inhibiting multiple anti-apoptotic proteins (inhibition of Mcl-1 with Bcl-2, Bcl-xL, or Bcl-2 and Bcl-xL). CLL cells with activation phenotype (CD5+/19+/69+) in patient PBMCs cultured without the agonist mix also showed tolerance to several apoptotic protein inhibitors ex vivo, and this was also effectively rescued with NF-kB inhibition or combination of apoptotic protein inhibitors ex vivo. These results suggest that the cancer cells with activation phenotype exist in vivo anddisplay multi-drug tolerant phenotype consistent with the drug tolerance induced by agonist mix ex vivo. In conclusion, several microenvironmental factors, particularly agonists of TLR9 and CD40, induce tolerance to IBR+VEN and cell proliferation in CLL/MCL, and response to these agonists is enhanced by combinatorial exposure. These agonists generate tolerance to several apoptosis-inducing agents beyond IBR+VEN. This microenvironment-induced multi-drug tolerance is mediated by NFkB dependent over-expression of multiple pro-survival proteins. Inhibitors of NFkB, or drug combinations targeting multiple pro-survival proteins, overcame multi-drug tolerance in agonist mix-treated samples and in CD69+CLL cells in patient PBMCs that represent multi-drug tolerant cells in vivo. Thus, more complete and durable responses might be achieved in MCL/CLL with therapies that target multi-drug tolerant persister cells. Disclosures Williams: Celgene: Consultancy, Research Funding; Kite: Consultancy; Juno: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy, Research Funding; Novartis: Research Funding; TG Therapeutics: Consultancy; Sandoz: Consultancy; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Takeda: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Verastem: Consultancy. Portell:AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy; TG therapeutics: Research Funding.

Description: Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (BTK), has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, IBR responses are incomplete in most cases, and of short duration for MCL and higher-risk CLL subtypes. We hypothesize that intrinsic resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be co-targeted with BTK inhibition to achieve deeper and more durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generated synergistic cytotoxicity with IBR in MCL lines as well as circulating leukemic B cells in 13/19 CLL and 4/5 MCL patient samples treated ex vivo (Jayappa KD et al. ASH 2015; Portell CA et al. ASH 2014). However, the sensitivity to VEN or IBR+VEN was highly variable among patient samples, and did not correlate with the diagnostic genetic lesions in the CLL/MCL cells or clinical characteristics of the patients. Here, we show that resistance to IBR+VEN can be induced by interaction with soluble factors found in the in vivo "macroenvironment" of the circulating cancer cells. To gain insight into changes in signaling pathways that might underlie mechanisms of drug synergy and resistance, we analyzed drug resistant MCL lines treated with IBR, VEN, and the combination by Reverse Phase Protein Arrays. We observed downregulation of PI3K-Akt, MAPK, JAK-STAT, and NOTCH signaling after IBR and IBR+VEN treatment, cleavage of caspases and PARP after VEN-treatment, and greatly enhanced cleavage of caspases and PARP after IBR+VEN treatment. A notable exception was significantly increased phosphorylation on p65 S536 of the NF-kB pathway at longer times after IBR+VEN treatment, indicating a possible role of NF-kB signalling in generating resistance to IBR and VEN in cells that survived treatment. To determine whether extrinsic factors in the cancer cell environment might be able to induce a drug-resistant phenotype, we co-cultured or pre-incubated PBMC with a diverse panel of stromal cells, cytokines, and other agonists. We found that the combination of soluble CD40L, IL-10, and CpG DNA (agonist mix) generated nearly complete resistance to IBR+VEN in CLL and MCL patient samples, with CpG DNA being the most effective single agent. Every sample treated with agonist mix displayed increased resistance to IBR+VEN drug combination, suggesting that responsiveness transcends genetic heterogeneity and reflects the developmental lineage of the cancer cells. We investigated whether the extrinsic agonists induce drug resistance by activating the NF-κB pathway, by analyzing nuclear localization of NF-kB transcription factors RelA and RelB using ImageStream and cell fractionation. We observed robust activation of alternative-NF-kB signaling in primary CLL and MCL cells treated with agonist mix, with little effect on canonical NF-κB. PKC, MAPK and PI3K-Akt signaling also showed evidence of activation by agonist mix. Agonist mix treatment also caused significant overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, but not Bcl-2. Inhibitors of NF-κB blocked RelB translocation and overexpression of Mcl-1, Bcl-xL and survivin. Inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame drug resistance induced by agonist mix. Inhibitors of the other activated pathways (MAPK, PI3K-Akt, PKC) did not block agonist-induced drug resistance at pharmacologically relevant concentrations. To determine whether extra-nodal agonists in the blood of patients could generate resistance to IBR and VEN, we analyzed drug cytotoxicity in CLL patient PBMCs cultured with autologous plasma. We found that autologous plasma was able to induce increased resistance to IBR+VEN in 3/7 CLL samples and this resistance was blocked by treatment with an NF-kB inhibitor. In conclusion, soluble agonists in the patient macroenvironment of circulating CLL/MCL cells can generate resistance to IBR+VEN by activating alternative-NF-kB signaling and over-expression of multiple anti-apoptotic proteins. Inhibitors of NF-kB or of the upregulated anti-apoptotic proteins overcame IBR+VEN resistance generated by these extrinsic factors. We suggest that survival signals generated by extra-nodal agonists in the patient macroenvironment generate drug resistance in CLL and MCL, and that improved responses could occur with interventions that block these survival signals. Disclosures Portell: AbbVie: Research Funding; Roche/Genentech: Research Funding; Infinity: Research Funding; Acerta: Research Funding. Wulfkuhle:Theranostics Health, LLC: Equity Ownership. Petricoin:Perthera, Inc.: Consultancy, Equity Ownership; Ceres Nanosciences, Inc.: Consultancy, Equity Ownership, Patents & Royalties; Avant Diagnostics, Inc.: Equity Ownership. Williams:University of Virginia: Employment; Janssen and Pharmacyclics: Research Funding.

Description: Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (Btk) and venetoclax (VEN), an inhibitor of Bcl-2 have been used in Chronic Lymphocytic Leukemia (CLL), but single agent responses to these drugs are often incomplete and not durable. We previously reported synergistic cytotoxicity for the IBR+VEN combination and have initiated a trial to test that combination (NCT02419560). However, we noted extensive variability in response to IBR+VEN between patient samples ex vivo, suggesting intrinsic tolerance even to this combination. CLL cells with an "activation" phenotype (CD5+/19+/69+) that occurs from interactions with the microenvironment were less sensitive to IBR+VEN, implicating the cancer microenvironment as an inducer of drug tolerance. This was supported by our finding that CLL cells display decreased sensitivity to IBR+VEN in co-culture with agonists and cells that emulate the cancer cell microenvironment. The combination of CpG-oligodeoxynucleotides (ODN), sCD40L, and IL10 ("agonist mix") induced near complete loss of sensitivity to IBR+VEN and upregulated Mcl-1 and Bcl-xL expression in CLL cells. However, Mcl-1 or Bcl-xL inhibitors alone were weakly effective in these cells, suggesting that exploitation of these targets requires drug combinations. Here we report a combination drug screen with an Mcl-1 inhibitor in a CLL microenvironment model ex vivo and novel drug combinations that overcome multi-drug tolerance. CLL patient PBMCs were cultured in the microenvironment model consisting of HK follicular dendritic cells, HUVEC, Jurkat T cells expressing CD40L, and CpG-ODN. Markers characteristic of activation and drug tolerance in vivo were assessed in CLL (CD5+/19+) cells using flow cytometry. We found increased expression of Ki67, CD69, and anti-apoptotic proteins Mcl-1 and Bcl-xL and decreased CXCR4 expression, as expected upon microenvironmental stimuli in vivo.Also, CLL cells from 2 IBR-treated patients were still responsive to agonist-induced drug tolerance, suggesting Btk inhibition may be less effective in blocking these microenvironmental stimuli in vivo. By comparing the ratio of anti-apoptotic proteins with cognate pro-apoptotic proteins, we noted targetable dependence of CLL cells for diverse pro-survival proteins (Mcl-1 and Bcl-xL) in our model. Flow cytometry analysis demonstrated that inhibitors of Mcl-1 (S63845), Bcl-xL (A1155643), or Bcl-2 (VEN) when used as single agents failed to induce significant apoptosis in CLL cells cultured in our model, suggesting induction of multi-drug tolerance. Drug combinations inhibiting Mcl-1 with Bcl-xL, Bcl-2, or Bcl-xL and Bcl-2 have effectively overcome multi-drug tolerance. As direct and simultaneous inhibition of multiple pro-survival proteins could be fatal to healthy cells, we carried out a Mcl-1-anchored combinatorial drug screen with inhibitors of other survival pathways in 3 CLL patient PBMCs cultured in our ex vivo model to identify drug combination(s) that induce selective toxicity in multi-drug tolerant CLL cells. Cytotoxicity was determined by analyzing cleaved PARP and dead cell staining in CLL and healthy T (CD3+/5+) cells by flow cytometry. Results showed that inhibitors of apoptotic proteins (A1155643, VEN, ABT737), IRAK4 (CA-4948, compound 26, and AS2444697), intracellular TLRs (chloroquine), Hsp90 (ganetespib), CDK (ribociclib), proteasome (bortezomib), Btk (IBR), AKT (MK2206), and HDAC (SAHA and panobinostat) or activator of PP2A (DBK1532 and NZ8061) were synergistically toxic with Mcl-1 inhibitor (S63845) in CLL cells. By comparing toxicity in CLL and healthy T cells, drug combinations targeting Mcl-1 with proteasome, IRAK4, TLRs, Btk, Hsp90, and CDK showed selective toxicity in CLL cells, indicating a potential therapeutic window for these combinations. Drug combinations targeting Mcl-1 with other apoptotic proteins were highly toxic to healthy T cells. In conclusion, CLL cells in our ex vivo model display characteristics of microenvironmentally-induced multi-drug tolerance. A similar phenotype was noted using post IBR therapy samples, suggesting IBR alone may not be effective in overcoming this multi-drug tolerance in vivo. Drug combinations targeting Mcl-1 and proteasome, IRAK4, TLRs, Btk, Hsp90, or CDK selectively overcame multi-drug tolerance in CLL cells. Thus, these combinations may be effective in patients showing intrinsic tolerance to multiple drugs. Disclosures Portell: TG therapeutics: Research Funding; AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy. Narla:University of Michigan: Patents & Royalties: Small molecule PP2A activators. Williams:Takeda: Research Funding; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Verastem: Consultancy; Juno: Consultancy; Sandoz: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Kite: Consultancy; Gilead: Consultancy, Research Funding.

Description: Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK. In Phase II studies of single-agent ibrutinib in MCL (Wang ML et al, NEJM 2013) and CLL (Byrd JC, et al, NEJM 2013) the overall response rate was 68% and 89% (CR, PR, and PR with lymphocytosis), respectively, with PR as the best response in the majority of patients. Thus, not all patients respond and complete responses are infrequent with single agent ibrutinib. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines using a focused drug panel (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy. Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 substantially induced apoptosis compared to each single agent alone (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL and normal donor samples, heterogeneity in response to the combination of ibrutinib and ABT-199 was seen. When evaluated by Bliss modeling, 5 of 9 CLL samples had a synergistic improvement in apoptosis with the combination with the other 4 having no change. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors. Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy. In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned. Disclosures Off Label Use: Pre-clinical data with ABT-199 for MCL and CLL, not FDA approved. Williams:Pharmacyclics, Janssen: Consultancy, Research Funding.

Description: The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin's Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017). Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis. Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D). To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C). In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Disclosures Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla:The Icahn School of Medicine at Mount Sinai: Patents & Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.