ALBERT

Description: BACKGROUND The prior Australasian Leukaemia and Lymphoma Group (ALLG) CLL5 Study showed dose-reduced oral fludarabine and cyclophosphamide plus rituximab (FCR3) was safe, tolerable and effective in fit elderly patients for front-line therapy for CLL. The German CLL11 Study showed chlorambucil plus obinutuzumab (Cbl+G) was superior to chlorambucil alone or with rituximab in unfit patients requiring initial therapy. We conducted a randomized study to assess the safety, tolerability, and efficacy of dose-reduced FC + obinutuzumab (G) (FC+G) versus Cbl+G in unfit (i.e. with comorbidity), elderly patients with CLL. METHODS Patients aged ≥65 years and considered "unfit" defined by co-morbidities using the Cumulative Illness Rating Scale [CIRS] ≥6 were eligible for the ALLG CLL7 study. Patients with any single organ system score ≥4 were excluded. Previously untreated patients with progressive CLL aged ≥65 and CIRS ≥6 were randomised to one of 2 therapy arms: (i) Chlorambucil 0.5mg/kg D1+15 p.o. + obinutuzumab ("G") (i.v. 1000mg/m2 cycle 1, Day 1, 8, 15, and 1000mg/m2 D1 cycles 2-6), or (ii) FC(rd)+G: F-24mg/m2 p.o. and C-150mg/m2 p.o. D1-3 + G (same schedule above) at 4 weekly intervals for planned 6 cycles. Early stopping for toxicity was mandated: treatment could be delayed for 2 weeks for grade 3+ toxicity, but if unresolved by 2 weeks, patients were taken off study. The primary end-point was grade 3+ non-hematological, and grade 4 hematological adverse events. Secondary objectives were overall response rate (ORR), complete remission (CR), partial remission (PR), progression-free survival (PFS) and overall survival (OS) and minimal residual disease (MRD) negativity. Final staging was performed between 2-3 months following final treatment cycle. RESULTS Patient characteristics Patient recruitment was terminated early due to poor recruitment. At the time of study closure, there were 32 patients, with 15 on Cbl+G and 17 on FC(rd)+G. The mean age was 74.2 years (range 66-85 years) with 23 females (71.9%) and 9 males (28.1%). The CIRS score was 6 in 4 patients (12.5%), 6-8 in 14 (43.8%), 8-10 in 11 (34.4%) and 〉10 in 3 (9.4%). Binet stage at registration was stage A 18.2%, B 27.3% and C 54.5%. Tolerability Both therapies were tolerable with 15/17 (88.2%) completing all 6 cycles of FC(rd)+G and 12/15 (92.3%) completing six cycles of Cbl+G. Toxicity Most toxicity was hematological and manageable. Grade 3/4 hematological toxicity was more common with FC(rd)+G than Cbl+G occurring in 60% with FC(rd)+G and 38.5% with Cbl+G (Table 1). There was one death due to progressive CLL on the FC(rd)+G arm. Response rate A complete remission (CR), confirmed by bone marrow (BM) trephine, was achieved in 86.6% of patients on FC(rd)+G versus (vs) 53.9% on Cbl+G, partial response (PR/nPR) in 1 (6.7%) on FC(rd)+G, and 6 (46.2%) on Cbl+G, and either stable or progressive disease (SD or PD) on 1 on FC(rd)+G, and nil on Cbl+G. BM MRD-negativity rates were 3/17 (20.0%) FC(rd)+G vs 1/15 (7.7%) Cbl+G (Table 2). CONCLUSION This randomized trial of dose-reduced FC(rd)+G vs Cbl+G in elderly patients aged ≥65 and with co-morbidities (CIRS ≥6) was terminated early due to poor recruitment. Due to the dose-reduced FC, and early stopping rule, treatment was safe and tolerable and most patients completed all 6 cycles of planned therapy. Grade 3/4 toxicity was mainly hematological and manageable, with higher rates of neutropenia with the FC (60%) vs Cbl (35.7%) backbone. FC(rd)+G compared to Cbl+G resulted a higher CR rate of 86.6%% versus 53.9%, and higher MRD-negativity (20% vs 7.7%). Progression-free and overall survival are being evaluated. Disclosures Badoux: Roche: Research Funding. Cull:Takeda Australia: Other: Travel Expenses; Amgen Australia: Other: Travel Expenses; AbbVie (Australia): Membership on an entity's Board of Directors or advisory committees. Tam:Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: BACKGROUND Trials of ibrutinib and idelalisib for Chronic Lymphocytic Leukemia (CLL) targeting Btk and PI3-kinase respectively, illustrate the potential of targeting components of the B-cell receptor (BCR) signalling pathway. Emerging evidence suggests that subgroups of CLL patients develop resistance to these agents. In particular late relapses on ibrutinib appear to be associated with a high frequency of acquired mutations in Btk and PLCg2. Identification of novel combination therapies or agents that target multiple molecules in key signaling pathways represents a rational approach in the development of novel treatment strategies. Pim (provirus integration site for Moloney murine leukemia virus) family proteins are proto-oncogenic and involved in B-cell development and lymphoid malignancies. They are highly conserved serine/threonine kinases and are overexpressed in CLL. Given the clinical efficacy of idelalisib and results of preclinical studies of the PIM kinase SGI-1776 [Chen et al., 2009], we sought to investigate the potential of simultaneous inhibition of PIM and PI3-kinase for CLL therapy. METHODS The effects of the novel inhibitor of PIM kinase, pPIMi, and the dual inhibitor of PIM/PI3-kinase, IBL-202 (both from Inflection Bioscience Ltd, Ireland), were investigated against panels of primary CLL patient samples and haematological lines. The in vitro efficacy of both agents was compared to that of SGI-1776 and idelalisib and studied under conditions involving CLL cell co-culture using CD40L-expressing fibroblasts that mimic the support offered by the CLL tumor microenvironment. RESULTS Inhibition of PIM kinase by pPIMi was as effective as SGI-1776, inducing a dose dependent decrease in CLL-cell viability. The rationale for dual inhibition of PIM and PI3-kinase was investigated by combining pPIMi with idelalisib and by calculation of combination indices (CI), where CI values of 〈 1 are indicative of synergy. In primary CLL patient samples (Figure 1) and 3 B-cell lines (Mec1, Ramos and Raji) the combination proved highly synergistic, with CI values of 0.07 + /- 0.03 and 0.11 +/- 0.07 respectively, for fractional cell killing effects of between 0.1 and 0.9. Consistent with our assessment of synergy between pPIMi and idelalisib, dual inhibition of PIM and PI3-kinase by IBL-202 was significantly more effective at inducing cell death than either pPIMi or idelalisib as single agents (Figure 2), evidenced by the significantly lower IC50 against CLL patient samples (7.47 µM, 43.12 µM, 1.23 µM for pPIMi, idelalisib and IBL-202 respectively). The rationale for targeting PIM and PI3-kinase using IBL-202 was further highlighted by culturing CLL cells in the presence of a CD40L-expressing fibroblast monolayer, which represents a model of the tumor microenvironment. CD40L fibroblast co-culture significantly increased CLL-cell survival under control conditions but also conferred resistance to the PIM inhibitor pPIMi. In contrast CLL cells cultured under these conditions were sensitive to IBL-202 in a dose dependent manner. CONCLUSION Inhibition of PIM and PI3-kinase is potently synergistic against CLL cells and is particularly effective under conditions that mimic the tumor microenvironment. Dual targeting of PIM and PI3-kinase by IBL-202 may represent a novel therapeutic strategy for CLL that minimizes the risk of acquired mutations associated with mono-molecular inhibition of BCR-signaling components. Figure 1. Synergy between pPIMi and idelalisib in primary CLL patient samples supports dual inhibition of PIM and PI3-kinase by IBL-202. Figure 1. Synergy between pPIMi and idelalisib in primary CLL patient samples supports dual inhibition of PIM and PI3-kinase by IBL-202. Figure 2. Dose response analyses of pan PIM inhibitor (pPIMi), idelalisib (CAL-101) and PIM/PI3-kinase inhibitor (IBL-202) against primary CLL patient samples (n = 9). Figure 2. Dose response analyses of pan PIM inhibitor (pPIMi), idelalisib (CAL-101) and PIM/PI3-kinase inhibitor (IBL-202) against primary CLL patient samples (n = 9). Disclosures O'Neill: Inflection Biosciences: Employment, Equity Ownership. O'Dwyer:Inflection Biosciences Ltd: Membership on an entity's Board of Directors or advisory committees. Mulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

Description: BACKGROUND: Immunochemotherapy (ICT) with fludarabine (F), cyclophosphamide (C) and rituximab (R) provides prolonged progression free (PFS) and overall survival (OS) in fit younger patients with CLL. The Australasian Leukaemia and Lymphoma Group (ALLG) CLL5 study previously showed FCR based therapy was safe, tolerable and effective in fit older CLL patients. We now assess the PFS and OS status by treatment arm and mutational status after a minimum of 5 years following final recruitment. METHODS: Previously untreated patients with progressive CLL aged ≥65 were randomised to one of three treatment regimens FR5, FCR3 and FCR5 (= full dose) as follows: (i) F 24mg/m2 po D1-5 + R (375mg/m2 C1, 500mg/m2 C2-6) iv D1 (FR5), (ii) F 24mg/m2 po and C 150mg/m2 po D1-3 + R iv D1 (FCR3) or (iii) F 24mg/m2 po+ C 150mg/m2 po D1-5 + R iv D1 (FCR5), all given at 4 weekly intervals for an intended 6 cycles. Early cessation of therapy was mandated for grade 3+ toxicity that delayed the next cycle 〉2 weeks on 〉2 occasions. RESULTS: The ITT population comprised 119 patients: 40 female and 79 male. Mean age was 71.6 years (range 64-83). The distribution by treatment arm was even with 38 patients on FR5, 41 on FCR3, and 40 on FCR5. As previously presented (ASH 2014), the overall response rate (ORR) was comparable at 95%, 95% and 97%, and the bone marrow confirmed complete remission (CR) rates 27%, 44% and 44% respectively. ORR and CR were not statistically significant. Toxicity was tolerable, and mainly hematological with neutropenia and thrombocytopenia. Early stopping due to toxicity occurred in 5.6%, 2.4% and 34% respectively, mainly hematological toxicity without complications. After a minimum of 5 years follow-up, no significant difference by OS (p=0.48) or PFS (p=0.93) (figure 1) was seen by treatment arm. Overall 51 patients (of 117 - 43.6%) died, hence the survival rate was 56.4%. Causes of death by treatment arm for FR5, FCR3 and FCR5 respectively were disease progression 4, 4, 1; Richter transformation 2, 0, 0; infection 2, 0, 7; and second malignancy 2, 4, 4. Of the 119 patients, 61 (51%) had data on immunoglobulin variable gene (IGHV) mutational status. In 33 (54%), IGHV were mutated (M-CLL) and 28 (46%) had unmutated IGHV (UM-CLL). The distribution by mutation status by treatment arm was even, with 10 each in FR5 and FCR3 and 13 in FCR5. Patients with M-CLL had a 61% lower risk of death and a significantly better PFS (p=0.0079; HR 0.39 [95% CL, 0.19 to 0.80]) than UM-CLL (M-CLL median PFS 110 months vs UM-CLL median PFS 48 months) (figure 2). CONCLUSIONS: Oral F(C)R therapy is generally safe and well tolerated in CLL patients aged ≥65 years requiring first-line treatment, when early stopping is utilized if prolonged toxicity is encountered; one third on full dose FCR5 stopped early with this rule, mainly with neutropenia. Response rates were high with ORR of 96% and CR rate of 56%. After a minimum of 5 years follow-up, OS and PFS outcomes by the treatment arms FR5 (full dose F, no C), FCR3 (40% FC dose reduction) and FCR5 (full-dose FCR) are essentially identical in this randomized dose de-escalation study. The median PFS overall was 53 months. CLL patients with M-CLL had a significantly superior PFS compared to UM-CLL. Disclosures Gill: Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau. Cull:Takeda Australia: Other: Travel Expenses; Amgen Australia: Other: Travel Expenses; AbbVie (Australia): Membership on an entity's Board of Directors or advisory committees. Simpson:Novartis: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Bristol-Myers Squibb: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Sanofi: Research Funding; BeiGene: Research Funding; Merck: Honoraria, Research Funding; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Roche: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Research Funding, TRAVEL, ACCOMMODATIONS, EXPENSES; MSD: Honoraria; Janssen: Honoraria, Research Funding. Tam:Pharmacyclics: Honoraria, Travel funding; Gilead: Honoraria; Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Roche: Honoraria; AbbVie: Honoraria, Research Funding; Roche: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria; Beigene: Honoraria, Other: Travel funding; Beigene: Honoraria, Other: Travel funding. Badoux:Roche: Research Funding.

Description: Background The B-cell receptor (BCR) signaling pathway and the pro-survival Bcl-2-family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Constitutive activity of the BCR signaling pathway and overexpression of Bcl-2 promote CLL-cell survival, proliferation and drug resistance. BCR-targeted therapies, most notably ibrutinib and idelalisib and the Bcl-2 inhibitor Venetoclax, demonstrate the potential of targeting these pathways. However, there is no evidence that these novel agents are curative in the event of relapse. Treatment options remain limited for patients treated with these agents, in particular those with TP53 lesions. In our recent study (Crassini et al., BJH 2018) we demonstrated efficacy of the PI3/PIM kinase inhibitor, IBL-202 (Inflection Biosciences, Ltd), against CLL cells. In the current study we investigated the effects of combining IBL-202 with Venetoclax on primary CLL cells and both wild-type and TP53 deficient OSU-CLL cells. Methods Primary CLL cells were co-cultured with CD40L-expressing fibroblasts. We established a TP53 knock-out OSU-CLL cell line (OSU-TP53ko) using the CRISPr-Cas9 system. Cell viability was assessed using the mitochondrial dye DilC1(5), propidium iodide and flow cytometry. Synergy between IBL-202 and Venetoclax was evaluated by determining combination indices (CI) using the Compusyn software. The effects of the drugs on cell cycle and proliferation of the OSU-CLL cell lines were assessed using propidium iodide or carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. The effects of the drugs on the migratory capacity of CLL cells were assessed by determining changes in CXCR4 expression and CLL-cell migration along an SDF-1a gradient. The mechanisms of action of the drugs were investigated by immunoblotting. Results IBL-202 and Venetoclax were highly synergistic against primary CLL cells co-cultured with CD40L-fibroblasts, with a CI of 0.4 at a fractional effect of 0.9 (Figure A and B). Synergy between the drugs was consistent with a significant (P 〈 0.05) reduction in the IC50 for both drugs. Synergy was also observed against wild-type (WT) and TP53ko OSU-CLL cells, with CI values of 〈 0.5 at fractional effects of 0.5 and 0.9. Synergy was consistent with significantly greater cytotoxic effects of the drugs in combination (Figure C. WT : P = 0.002 and TP53ko : P = 0.002). IBL-202 and Venetoclax in combination induced cell cycle arrest and slowed the proliferation of both cell lines. Immunoblotting of primary CLL cells showed IBL-202, alone and in combination with Venetoclax, inhibited AKT phosphorylation and reduced the expression of Mcl-1 and Bcl-xL. A greater than additive effect of IBL-202 and Venetoclax was observed on the migratory capacity of CLL cells, reducing the number of cells migrating towards SDF1-a. The effects of the drugs on cell migration were consistent with reduced expression of CXCR4. Conclusions The synergy we observed between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53ko OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for poor risk CLL disease. Figure A and B - Synergy of IBL-202 and Venetoclax against primary CLL cells. Figure C - Cytotoxic effects of IBL-202 in combination with Venetoclax against OSU and OSU-TP53ko CLL cells Disclosures O'Dwyer: Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Description: Background and Aim: Processing and cryopreservation is known to reduce the viability and absolute number of CD34+ haematopoetic stem cells (HSCs). DNA damage and subsequent apoptosis of stem cells may occur as a result of prior treatment regimens or from the accumulation of reactive oxygen species (ROS) during the freeze/thawing process. In our recent study we demonstrated that two distinct populations of HSCs are present in all cryopreserved patient samples, based on their ROS expression; these we defined as ROShigh and ROSlow. The significant correlation between the percentage of ROShigh HSCs in the graft and neutrophil recovery following transplant shown in our study suggests that some degree of ROS accumulation may play an important role in promoting neutrophil engraftment following autologous HSC transplantation (AHSCT) (Bai et al 2018). The current study examined the association between ROS levels and DNA damage in cryopreserved HSCs and whether these correlated with time to neutrophil and platelet engraftment following AHSCT. Methods: Cryopreserved HSC samples from 51 patients who underwent AHSCT were studied. HSC's were defined as CD45+/CD34+. DNA damage and intracellular ROS levels were assessed using an antibody against the phosphorylated form of histone H2Ax (γH2Ax) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Data acquisition and analysis were performed by flow cytometry. Results: We observed a proportion of HSC in each sample that expressed elevated levels of gH2Ax. The median percentage of γH2Ax expressing CD34+ cells was 54.6% (range 3.4 - 98.4%). We observed a strong correlation between ROShigh and γH2Ax levels in terms of the dose of cells/kg infused (p 〈 0.001, r = 0.72). The ratio of ROShigh/γH2Ax-expressing HSCs was inversely associated with delayed neutrophil engraftment (p = 0.03, r = -0.31). The cohort was divided into group 1 and group 2 based on ROShigh/γH2Ax ratios of 〉1 (n = 32) and ≤1 (n = 18) respectively. Median days to neutrophil engraftment were 12 (range 9-18), in group 1 and 15 (range 9-28) in group 2, (p = 0.007). Median days to platelet engraftment were 17 (range 10-28) in group 1 and 21.5 (range 14-55) in group 2, (p = 0.001). Median days to platelet count of 〉50 (plt50) was 18.5 (range 12-28) in group 1 and 22 (range 16-75) in group 2, (p = 0.001). In group 2, significant correlations were noted between the ROShigh/γH2Ax ratio and time to neutrophil engraftment (r = -0.54, p=0.025), plt20 (r = -0.64, p=0.004) and plt50 (r = -0.7, p = 0.002). Conclusion: Our data suggest that DNA damage in CD34+ and accumulation of ROS may impact upon the time to engraftment of HSCs. Calculating a ratio of ROShigh/γH2Ax expression may be useful in predicting engraftment time following autologous transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Background Chemo-immunotherapy remains the backbone of therapy for patients with chronic lymphocytic leukaemia (CLL), with evidence pointing towards long term remission and even cure in patients with mutated IGHV status receiving this therapy frontline (1). However, relapse is common especially in those harbouring abnormalities in TP53 or ATM, unmutated IGHV status or a complex karyotype (2). It has become increasingly apparent that the bone marrow (BM) and lymph node (LN) play important roles in promoting the survival and proliferation of CLL cells. Signalling pathways triggered by interactions within these niches, such as the B cell receptor (BCR) pathway, and intracellular proteins such as Bcl-2 are vitally important in the biology of CLL. Novel therapeutic agents, such as ibrutinib, which target components of the BCR pathway, and the Bcl-2 inhibitor venetoclax, have demonstrated the potential of targeted therapies in CLL (3, 4). Novel therapeutic approaches must target the proliferative, drug-resistant compartments of disease within these microenvironments. The NanoString® nCounter platform enables mRNA profiling of archival samples, including formalin-fixed, paraffin-embedded tissue (FFPE). We have previously demonstrated the utility of this technology by comparing the mRNA expression profile of CLL cells derived from the peripheral blood (PB), archival BM and LN tissue as well as PB-derived CLL cells following in vitro co-culture with a human stromal cell line under either normoxic or hypoxic conditions. Here we present an update on our previous work with increased sample numbers in each of the tissues or culture conditions. Methods RNA was extracted from FFPE BM trephines (n = 5) and LN sections (n = 5), using the QIAGEN RNeasy FFPE Kit. All biopsies analysed were comprised of 〉 80 % lymphocytes, as determined by microscopic review. RNA from PBMC fractions (n = 5) was isolated either immediately or following co-culture with HS5 stromal cells for 24 h under normoxic (n = 5) or hypoxic (n = 5) conditions using the QIAGEN RNeasy Mini Kit. RNA from all preparations was quantified using a NanoDrop™ spectrophotometer. A total of 200ng of FFPE-derived RNA and 100ng of PBMC-derived RNA was analysed per sample on the NanoString® platform using a 260 gene panel. Three-fold changes in mRNA expression were considered significant. Results Of the 260 genes profiled, 89 were upregulated in the BM samples and 52 in the LN samples compared to expression in PB-derived CLL cells. Changes were seen in genes encoding for proteins involved in chemotaxis (CXCL9), the regulation of apoptosis (BCL2L1), surface receptors (FLT3) and genes associated with intracellular signalling, metabolism and cell division. 35 genes were downregulated in the LN samples and 31 in the BM samples. These changes were seen in genes coding for surface receptors (ROR1 and CXCR4), genes coding for intracellular signalling proteins (RAF1) and genes coding for transcription factors (JUN and FOS). Co-culture of PBMCs with HS5 cells induced similar changes to those observed in our comparison of the PBMCs and BM samples; genes coding for 61.5% and 50.0% of the mRNA expression changes observed in the LN were observed in PBMCs cultured under normoxic and hypoxic conditions respectively. A similar comparison of the BM samples identified concordant changes in expression of 46.5% and 39.2% of genes under normoxic and hypoxic conditions respectively. Importantly, changes observed in genes coding for the anti-apoptotic protein MCL1, the surface receptors CXCR4 and ROR1 and the transcription factors ATF, FOS and JUN were consistent across samples from LN, BM and the in vitro model. In summary, we have utilised the NanoString® nCounter platform to profile PB, BM and LN-derived CLL cells and have identified panels of genes that are either up or down-regulated in cells derived from these microenvironments. Furthermore, the high concordance between RNA changes in the in vitro model and the primary tissue suggest the HS5 co-culture system mimics aspects of the tumour microenvironment. These data provide a better understanding of how CLL cells populate and proliferate in the tumour microenvironment and may lead to novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Description: Background The PI3-kinase signaling pathway and the Bcl-2-family of proteins play crucial roles in regulating the survival and proliferation of chronic lymphocytic leukemia (CLL) cells in the bone marrow and lymph nodes. Trials of ibrutinib, idelalisib and venetoclax illustrate the potential of targeting the B-cell receptor (BCR) signaling pathway and Bcl-2, however disease relapse is still common. Several pre-clinical studies and on-going clinical trials [Rogers et al., 2018, Jain et al., 2019] suggest that combinations of BCR inhibitors with venetoclax may be an effective treatment strategy for CLL patients with high risk disease. We sought to investigate the effects of combining idelalisib or the AKT inhibitor MK2206 with venetoclax against CLL cells under in vitro conditions that mimic the tumor microenvironment. Methods Primary CLL cells were co-cultured with CD40L-expressing mouse L-fibroblasts. Cell viability was assessed using the mitochondrial membrane potential dye DilC1(5), propidium iodide and flow cytometry (n = 6). Synergy between idelalisib or MK2206 and venetoclax was evaluated by calculating combination indices (CI) using the Compusyn software. The mechanisms of action of the drugs and synergies between the drugs were investigated by immunoblotting (n = 6). Results Venetoclax was highly synergistic in combination with idelalisib or MK2206 against CLL cells co-cultured with CD40L-fibroblasts, with CI values of 0.2 and 0.5 at a fractional effect of 0.9, respectively (Figure 1). This synergy was consistent with a significant (P 〈 0.05) reduction in the IC50 for venetoclax, idelalisib and MK2206. Immunoblotting suggests that MK2206, as a single agent or in combination with venetoclax, was more effective than idelalisib in inhibiting the phosphorylation of AKT and NF-κB. Both MK2206 and idelalisib as single agents and in combination with venetoclax significantly reduced expression of Mcl-1 and Bfl-1, two pro-survival members of the Bcl-2 family of proteins in primary CLL cells co-cultured with CD40L-fibroblasts. Conclusions The synergy observed, which was associated with a significant decrease in the IC50s for idelalisib and MK2206, may mitigate some of the toxicities associated with PI3-kinase pathway inhibitors. Comparison of the two PI3-kinase-pathway inhibitors suggests that MK2206 may be more effective than idelalisib at blocking BCR-mediated signaling as a single agent and in combination with venetoclax. The mechanisms underlying the synergy include down-regulation of expression of Bcl-2 family proteins that are not targeted by venetoclax as a single agent. The data presented support the rationale for on-going and future clinical trials of combination therapies incorporating a PI3-kinase inhibitor with venetoclax for the treatment of high risk CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Description: Introduction Despite the revolution in the treatment of chronic lymphocytic leukemia (CLL) over the past decade with the introduction of novel inhibitors targeting the B-cell receptor (BCR) signaling pathway and the Bcl-2 family of proteins, relapse is still common. Recent studies suggest that imipridones, a novel class of small molecule agents that attenuate mitochondrial respiration and modulate an immune response against cancer cells, may be an effective treatment option for several difficult to treat cancers. We investigated the effects of the imipridone, ONC-212 (I-39, first published by Nanjing Gator Meditech), as a potential therapeutic strategy for CLL using the OSU-CLL cell line and a modified OSU-CLL line in which TP53 was stably knocked out and primary CLL cells cultured under conditions that mimic the tumour microenvironment (TME). Methodology Primary CLL cells were co-cultured with CD40L-expressing fibroblasts to mimic aspects of the TME. The cytotoxicity of ONC-212 was assessed using the mitochondrial dye DiIC1(5), propidium iodide and flow cytometry. The effects of the drug on the adhesive and migratory capacity of primary CLL cells were evaluated using antibodies against CD49d, CXCR4 and an in vitro migration assay using stroma-derived factor 1a (SDF1-α). Changes in protein expression were assessed by immuno-blotting. The effects of ONC-212 on the cell cycle and proliferation were assessed using the OSU-CLL cell line. OSU-CLL cells were modified using the CRISPr-Cas9 technology to be TP53 deficient (OSU-TP53ko). The proportion of cells in each cycle phase was determined using propidium iodide and flow cytometry. Cell proliferation rates were determined using carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. Results ONC-212 induced apoptosis in a dose-dependent manner in primary CLL cells cultured in medium alone or in contact with CD40L-fibroblasts (Figure 1); the IC50 values were 72.97 nm +/- 1.45 nM and 472 +/- 2.04 nM, respectively. OSU-CLL and OSU-TP53ko cells were also sensitive to ONC-212, although the TP53 deficient line was less sensitive than OSU-CLL(Figure 1). IC50 values for the cell lines were 22 +/- 1.37 nM (OSU-CLL) and 48 +/- 3.25 nM (OSU-TP53ko). ONC-212 induced cell cycle arrest of the OSU-CLL and OSU-TP53ko lines at the G1/S phase transition. This effect was concomitant with a significant reduction in the proliferation of both lines. ONC-212 significantly down-regulated expression of the adhesion molecule CD49d and the G-coupled protein receptor CXCR4 on primary CLL cells. Down-regulation of CXCR4 translated into a decrease in the migratory capacity of CLL cells along an SDF1-α gradient. Immunoblotting suggested the mechanisms of action of ONC-212 include inhibition of ERK1/2-MAPK, a decrease in the Bcl-2/Bax ratio and upregulation of the pro-apoptotic Puma and Bak proteins. Conclusions ONC-212 is highly effective against CLL cells at nanomolar concentrations, against cells cultured under conditions that mimic aspects of the TME and against TP53-deficient cells. ONC-212 has cytotoxic effects, induces cell cycle arrest, slows proliferation and inhibits the mechanisms by which CLL cells migrate to and are retained within the TME. ONC-212 inhibited signaling downstream of the BCR and induced a pro-apoptotic 'tipping' of the balance in expression of BCl-2 family proteins. These data suggest ONC-212 may represent an effective treatment for CLL, particularly for patients who have high risk, relapsed/refractory disease associated with loss or mutation of TP53. Disclosures No relevant conflicts of interest to declare.