Targeting Btk Research Articles

Investigating the molecular mechanisms underlying the co-occurrence of Parkinson’s disease and inflammatory bowel disease through the integration of multiple datasets

Parkinson’s disease (PD) and inflammatory bowel disease (IBD) are chronic diseases affecting the central nervous system and gastrointestinal tract, respectively. Recent research suggests a bidirectional relationship between neurodegeneration in PD and intestinal inflammation in IBD. PD patients may experience gastrointestinal dysfunction over a decade before motor symptom onset, and IBD may increase the risk of developing PD. Despite the “gut-brain axis” concept, the underlying pathophysiological mechanisms of this potential association remain unclear. This study aimed to investigate the biological mechanisms of differentially expressed genes in PD and IBD using bioinformatics tools, providing novel insights into the co-diagnosis and treatment of these diseases. We constructed a gene marker for disease diagnosis and identified five important genes (BTK, NCF2, CRH, FCGR3A and SERPINA3). Through nomogram and decision tree analyses, we found that both the IBD and PD required only the expression levels of BTK and NCF2 for accurate discrimination. Additionally, small molecule drugs RO-90-7501 and MST-312 may be useful for the treatment of both IBD and PD. These findings offer new perspectives on the co-diagnosis and treatment of PD and IBD, and suggest that targeting BTK may be a promising therapeutic strategy for both diseases.

Combined Inhibition of CDK2 and BCL2 Overrides Resistance to Targeting BTK and CDK4/6 in Mantle Cell Lymphoma Therapy

Mantle cell lymphoma (MCL) remains incurable due to the development of drug resistance, despite the plethora of therapies available. Each successive treatment failure is associated with a more rapidly proliferating disease and fewer treatment options. Understanding the drug-resistance mechanism and developing effective, well-tolerated, new therapies for MCL are urgently needed. Unrestrained proliferation of MCL cells is mainly driven by aberrant cyclin D1 expression and dysregulated CDK4 activity that promotes cell cycle progression from G1 to S. In our phase 1 clinical trial inhibiting CDK4/6 with palbociclib and BTK with ibrutinib (PALIBR) in recurrent MCL, the complete response rate (CR) was 42% compared to 21% in response to ibrutinib alone, despite a comparable 67% overall response rate. Moreover, 5 patients remain on therapy (2 CR and 3 PR) for ~ 8 years, suggesting that CDK4/6 inhibition deepens and prolongs the clinical response to BTK inhibition in MCL. However, resistance emerges in some patients. To elucidate the underpinnings, we undertook integrative longitudinal genomic analysis of sequential tissue and blood specimens (n=53) of 27 MCL patients before, during therapy and on progression, and 4 treatment-naïve MCL patients and 4 normal subjects as controls. Whole exome and whole transcriptome sequencing of freshly isolated MCL cells and B cells revealed that resistance to PALIBR is rooted in homozygous deletion of RB1 (direct target of CDK4/6) or compound copy number variation (cCNV) - concurrent hemizygous loss of RB1 and CDKN2A (p16, CDK4/6 inhibitor) and gain of CDK4. In other resistant patients, the RB protein is silenced by translational termination mutation. Single-cell RNA-sequencing (scRNA-seq, 210,000 cells) of corresponding PBMCs using a unique in-house MCL RNA reference library reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative, expanding with disease progression. RNA velocity analysis further uncovered that C2 fuels the transition to C4 with disease progression, whereas C3 is a terminal node (Fig 1). In C2 and C3 MCL cells, CDK2 is highly elevated, indicating evasion of CDK4/6 inhibition, and PIK3IP1 (negative PI3K regulator) is silenced, suggesting that compensatory PI3K activation in BTKi resistance (Chiron et al, Cancer Discovery 2014) is secondary to loss of cell cycle control. BCL2 is markedly elevated in C2, greater in C3 MCL cells along with increases in anti-apoptotic BCL2L1(BCL-XL) and BCL-2A1 and decreases in pro-apoptotic PMAIP1 (NOXA) expression. This suggests combined CDK2 and BCL2 inhibition as a rational approach to override CDK4/6i and BTKi resistance. Indeed, combined inhibition of CDK2 with PF-07104091 and BCL2 with venetoclax (PF-VEN) cooperatively impaired growth and killed ibrutinib-resistant MAVER-1 cells, more effectively in the isogenic derivative MAVER-1R cells which we generated to recapitulate cCNV in PALIBR resistance - depletion of RB protein through translational termination mutation (W99, AAF 75%) in RB1, loss of CDKN2A and gain of CDK4. Mechanistically, MAVER-1R cells are accelerated in G1 to S progression, based on elevation of CDK2 and TK1 expression and cell cycle profiling. They are intrinsically apoptotic and vulnerable to BCL2 inhibition due to a marked reduction in cells expressing anti-apoptotic BCL2L1 and IRF4, and universal expression of pro-apoptotic PMAIP1 (NOXA). Importantly, combined CDK2 and BCL2 inhibition synergistically kills MCL cells of a patient resistant to PALIBR ex vivo. In summary, by longitudinal genomic analysis, we have provided the first evidence that 1) resistance to CDK4/6i and BTKi stems from MCL intrinsic cCNV; 2) MCL cells comprise 4 major transcriptomically distinct clusters; cluster 2 expressing copious CDK2 and BCL2 is pivotal in fueling proliferation of MCL cells in resistance; 3) cluster 3 cells also accumulate with resistance due to longevity; 4) combined inhibition of CDK2 and BCL2 overrides resistance to CDK4i and BTKi, in MCL cell lines and primary MCL cells from PALIBR resistant patient. Combined inhibition of CDK2 and BCL2, therefore, represents a mechanism-based strategy to overcome CDK4/6i and BTKi resistance in MCL therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 5438: FCN-589, a novel, potent and selective BTK inhibitor, targets BTK C481S for the treatment of B-cell malignancies

Abstract Bruton’s tyrosine kinase (BTK) is a member of the Tec family of non-receptor tyrosine kinases and plays critical roles in B-cell receptor (BCR) signaling. BTK is often overexpressed in B-cells of patients with hematological malignancies. BTK inhibition blocks BCR signals, prevents B-cell activation and malignant B-cell growth, and induces apoptosis. Early irreversible BTK inhibitors such as ibrutinib, which covalently binds to the cysteine residue (C481) of BTK, are effective against B-cell malignancies and have received approval for various B-cell lymphomas and leukemia including mantle cell lymphoma and chronic lymphocytic leukemia, but their effectiveness is often limited by toxicities from off-target inhibition of the Tec (e.g., ITK/TEC) and EGFR family (e.g., EGFR/HER2/HER4). Acquired resistance mutations in the C481 binding site are known to cause clinical resistance to irreversible BTK inhibitors, which accounts for over half of progression in patients received ibrutinib. There is thus unmet medical need for a novel BTK inhibitor with improved safety profile efficacious against C481 mutation. Here we introduce FCN-589, a novel and orally active next-generation BTK inhibitor. FCN-589 shows potent kinase activities against wild-type (WT) BTK and the most common resistant mutation BTK C481S with single-digit nanomolar IC50 and improved selectivity over clinically significant off-targets such as TRK and ITK compared with nemtabrutinib (MK-1026, ARQ 531), another BTK C481S inhibitor in development. FCN-589 exhibits remarkable anti-proliferating potency against a panel of human B-cell malignancies-derived cancer cell lines. FCN-589 is over 30x more potent against follicular lymphoma cells (DOHH2) and activated B-cell subset of diffuse large B-cell lymphoma (DLBCL) cells (OCI-LY10 and Ri-1), and about 4x more potent against germinal center B-cell like subset of DLBCL cells (SU-DHL-4) compared with ARQ 531. In HEK-293 cell line ectopically expressing WT or BTK C481S, FCN-589 markedly inhibits p-BTK expression with superior activity compared with ARQ 531. Consistently, FCN-589 shows significant and superior anti-tumor activities in mice xenograft models derived from human non-Hodgkin’s lymphomas compared with ARQ 531 and ibrutinib. In non-clinical studies, FCN-589 exhibits good pharmacokinetic (PK) and safety properties. In particular, FCN-589 has longer T1/2 in both rats and dogs, displays improved CYP450 inhibition profile, and is less plasma protein bound compared with ARQ 531. Overall, FCN-589 is a novel next-generation BTK inhibitor targeting WT and BTK C481S. FCN-589 possesses potent in vitro and in vivo anti-cancer activities with better selectivity against clinically significant off-targets and exhibits favorable PK and safety profiles compared with ARQ 531. FCN-589 has potential to be a more effective and safer option for patients with B-cell malignancies. Citation Format: Shu Lin, Haohan Tan, Qihong Liu, Yunling Wang, Bin Liu, Zhifu Li, Zhifang Chen, Xianlong Wang, Xingdong Zhao, Lihua Jiang, Xilei Wang, Tao Cheng, Jiashu Zhou, Weibo Wang. FCN-589, a novel, potent and selective BTK inhibitor, targets BTK C481S for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5438.

BTK-PROTAC Targeting BTK Degradation as a New Weapon for the Treatment of B-Cell Lymphomas

BTK-PROTAC Targeting BTK Degradation as a New Weapon for the Treatment of B-Cell Lymphomas

A Phase I Study Of The Oral Btk Inhibitor ONO-4059 In Patients With Relapsed/Refractory and High Risk Chronic Lymphocytic Leukaemia (CLL)

IntroductionThe B-cell receptor (BCR) pathway plays a central role in signal transduction pathways that regulate survival, activation, proliferation and differentiation of B-lineage lymphoid cells. Bruton's tyrosine kinase (Btk) is a critical kinase in BCR signal transduction and recent studies support that targeting Btk is effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC50 in the sub-nmol/L range that has demonstrated anti-tumour activity in several pre-clinical models (Yasuhiro T et al, AACR 2013). MethodsThis Phase I study was initiated to determine the safety, tolerability, dose-limiting toxicity (DLT), pharmacokinetics and pharmacodynamics of ONO-4059 given as monotherapy to patients with relapsed/refractory, high risk CLL for whom no therapy of higher priority was available. In this safety-driven, dose-escalating 3+3 design, ONO-4059 was administered as an oral, daily dose (flat dose) given continuously initially for up to 6 months, with the option of additional dosing up to 2 years. We present the safety and efficacy data on 16 evaluable patients, median age 67 yrs (range 40-77), with high risk cytogenetics (17p [38% (6/16)] or 11q [19% (3/16)], 12/16 displaying unmutated IgVH status, 8/16 displaying TP53 mutation), median baseline tumour burden 4,534mm2 [461-14,492 mm2]. Patients had received a median of 3 prior therapies [2-7], including fludarabine (16/16) and rituximab-containing therapy (13/16). Baseline median haematology parameters included hemoglobin 11.3 g/dL [8.5 -15.2], WBC 56.02 x 109/L [2.06-394.74], platelets 95.5 x 109/L [23-173], lymphocytes 50.6 x 109/L [0.5-390], neutrophils 2.46 x 109/L [0.5-11.1]. Patients were treated with ONO-4059 at doses ranging from 20mg-320mg (cohorts 1-5) and the study is currently ongoing with additional dose escalation cohorts to be completed. ResultsONO-4059 was found to be well tolerated, with no dose limiting toxicities (DLTs). A total of 15 ONO-4059-related Grade 1 (n=10) and 2 (n=5) adverse events were reported in 8 out of 16 patients, including one event of diarrhea (G2) and two events of rash (G1, G2). ONO-4059-related G3 or G4 haematological toxicities were infrequent and independent of dose; neutropenia (n=3 [G4 x1, G3 x 2]), febrile neutropenia (n=1 [G3]) occurring in 2 patients at the 20mg dose level only. Only one Grade 3 ONO-4059-related non-haematological event (pyrexia) was reported. Infection rate was low with only one event of bronchitis reported (G2, related). Three ONO-4059-related SAEs were reported in two patients (febrile neutropenia [G3] and pyrexia [G3] in one patient [20 mg]; rash [G2] in another [80 mg]). The pharmacokinetics of ONO-4059 reflects rapid absorption and elimination, a half-life of ∼6 hours, a dose dependent increase in exposure with no accumulation of ONO-4059 exposure and low inter- or intra-patient variability; with Btk occupancy in peripheral blood (as measured by phosphorylated Btk) being maintained for at least 24 hours across all dose levels.Responses have occurred across all dose levels. All patients experienced rapid reductions in lymphadenopathy observed early in treatment, between D1-D28 of the first cycle, accompanied by an increase in absolute lymphocyte count in some, but not all patients. Best overall response rate according to IWCLL criteria (including modified PR with lymphocytosis) was 70% [based on CT-scan and P/E for 7/10 evaluable patients]; with 2 PR, 5 PR with lymphocytosis (For 7 responding patients, median reduction of lymph nodes was 67% [Range 61%-79%]), 2 SD, 1 patient PD (possible Richter's). All patients showed an improvement in haematological parameters after ≥3 months on treatment. Patients (ONO-4059 20mg [1pt] and 40mg [1pt] respectively) who had grade 3 thrombocytopenia at study baseline had a significant improvement in platelet count (>100 x 109/L) with ONO-4059 treatment. To date, 15 of 16 patients remain on treatment with a median progression-free survival of 99 days [Range 8-268].In conclusion, ONO-4059 is a highly potent and selective oral Btk inhibitor with a favourable safety profile along with promising efficacy in this heavily pre-treated population, with responses observed in relapsed, refractory and 17p deleted patients. Disclosures:Salles:Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria. Karlin:Janssen: Honoraria; Celgene: Honoraria. Morschhauser:ONO Pharma: Honoraria; Roche: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria. Dyer:ONO Pharma: Honoraria, Research Funding. Hutchinson:ONO Pharma: Research Funding. Fegan:ONO Pharma: Honoraria. Cartron:ONO Pharma: Honoraria. Knurowski:ONO Pharma: Consultancy. Saunders:ONO Pharma: Consultancy; Pharmacyclics: Consultancy. Wright:ONO Pharma: Consultancy. Honda:ONO Pharma: Employment. Mazur:ONO Pharma: Consultancy. Yoshizawa:Ono Pharma: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment. Birkett:Ono Pharma UK: Employment.

Transcriptomic Analysis of Inflammatory Cardiomyopathy Identifies Molecular Signatures of Disease and Informs in silico Prediction of a Network-Based Rationale for Therapy.

Inflammatory cardiomyopathy covers a group of diseases characterized by inflammation and dysfunction of the heart muscle. The immunosuppressive agents such as prednisolone, azathioprine and cyclosporine are modestly effective treatments, but a molecular rationale underpinning such therapy or the development of new therapeutic strategies is lacking. We aimed to develop a network-based approach to identify therapeutic targets for inflammatory cardiomyopathy from the evolving myocardial transcriptome in a mouse model of the disease. We performed bulk RNA sequencing of hearts at early, mid and late time points from mice with experimental autoimmune myocarditis. We identified a cascade of pathway-level events involving early activation of cytokine and chemokine-signaling pathways that precede leucocyte infiltration and are followed by innate immune, antigen-presentation, complement and cell-adhesion pathway activation. We integrated these pathway events into a network-like representation from which we further identified a 50-gene subnetwork that is predominantly induced during the course of autoimmune myocardial inflammation. We developed a combinatorial attack strategy where we quantify network tolerance to combinatorial node removal to determine target-specific therapeutic potential. We find that combinatorial attack of Traf2, Nfkb1, Rac1, and Vav1 disconnects 80% of nodes from the largest network component. Two of these nodes, Nfkb1 and Rac1, are directly targeted by prednisolone and azathioprine respectively, supporting the idea that the methodology developed here can identify valid therapeutic targets. Whereas Nfkb1 and Rac1 removal disconnects 56% of nodes, we show that additional removal of Btk and Pik3cd causes 72% node disconnection. In conclusion, transcriptome profiling, pathway integration, and network identification of autoimmune myocardial inflammation provide a molecular signature applicable to the diagnosis of inflammatory cardiomyopathy. Combinatorial attack provides a rationale for immunosuppressive therapy of inflammatory cardiomyopathy and provides an in silico prediction that the approved therapeutics, ibrutinib and idelalisib targeting Btk and Pik3cd respectively, could potentially be re-purposed as adjuncts to immunosuppression.

Targeting the NLRP3 Inflammasome via BTK.

The NLRP3 inflammasome represents a critical inflammatory machinery driving pathology in many acute (e. g., myocardial infarction or stroke) and chronic (Alzheimer's disease, atherosclerosis) human disorders linked to the activity of IL-1 cytokines. Although the therapeutic potential of NLRP3 is undisputed, currently no clinically approved therapies exist to target the NLRP3 inflammasome directly. The recent discovery of BTK as a direct and positive regulator of the NLRP3 inflammasome has, however, raised the intriguing possibility of targeting the NLRP3 inflammasome via existing or future BTK inhibitors. Here, I review the mechanistic basis for this notion and discuss the molecular and cellular role of BTK in the inflammasome process. Specific attention will be given to cell-type dependent characteristics and differences that may be relevant for targeting approaches. Furthermore, I review recent (pre-)clinical evidence for effects of BTK inhibitors on NLRP3 activity and highlight and discuss open questions and future research directions. Collectively, the concept of targeting BTK to target NLRP3-dependent inflammation will be explored comprehensively at the molecular, cellular and therapeutic levels.

Targeting BTK in CLL: Beyond Ibrutinib.

While the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL), current limitations include off-target toxicities and the development of resistance. In this review, we summarize the emerging data for alternative BTKi. Second-generation BTKi include acalabrutinib, zanubrutinib, and tirabrutinib which offer greater BTK selectivity. While these agents may limit off-target toxicity, they do not overcome common mechanisms of ibrutinib resistance. Reversible BTKi including vecabrutinib and LOXO-305 inhibit BTK in the presence of C481S mutation, and non-selective reversible BTKi, including ARQ-531, may retain activity despite mutations within PLCG2. Early-phase studies are underway to establish the clinical efficacy and toxicity of these agents. A randomized trial of ibrutinib versus acalabrutinib is ongoing, and acalabrutinib may be an option for ibrutinib-intolerant patients. Results from ongoing trials of alternate BTKi will help to define their role in CLL therapy as single agents or in combination therapy.

A good response of refractory mantel cell lymphoma to haploidentical CAR T cell therapy after failure of autologous CAR T cell therapy

BackgroundThe aggressive form of Mantle cell non-hodgkin B cell lymphoma (MCL) has a dismal prognosis. Dual targeting BTK and BCL2 with ibrutinib and venetoclax has improved outcomes in MCL patients who were predicted not to respond to conventional therapy, but it is unlikely to be curative. Chimeric antigen receptor-modified T (CAR T) cells exhibit very effective function in elimination of relapsed/refractory B-cell lymphoid malignancies, we investigated their use in a patient with relapsed MCL.Case presentationHere, we report a case of a refractory MCL in a patient who had relapsed after conventional chemotherapy and autologous CAR T cell therapy. The patient received multiple molecularly targeted therapies, including targeting BTK and BCL2, and haplo-identical CAR T (haplo-CAR T) cells from her daughter without previous allo-hematopoietic stem cell transplantation. Haplo-CAR T cells could effectively proliferate in vivo and had a clinically significant antitumor activity without serious side effects. The patient achieved a partial remission, with minimal residual disease.ConclusionsThis case suggests that haplo-CAR T cell therapy can be effective in controlling lymphoma that failed to respond to autologous CAR T cell therapy and overcome limitation of autologous CAR T cells, thus may be one possible regimen before the era of off-the-shelf “universal” CAR T cell therapy.Trial registrationChiCTR-OPN-16008526. http://www.chictr.org.cn/showproj.aspx?proj=13798; ChiCTR1800019385. http://www.chictr.org.cn/showproj.aspx?proj=32805; ChiCTR1800019449. http://www.chictr.org.cn/showproj.aspx?proj=32778.

Concurrent treatment with two B-cell receptor pathway inhibitors

The prognosis of B-cell lymphoid malignancies has improved after the implementation of ibrutinib and idelalisib: two orally-available drugs targeting the B-cell receptor signalling pathway. Ibrutinib is a Bruton tyrosine kinase inhibitor (BTKi) approved for chronic lymphocytic leukaemia, mantle cell lymphoma, and Waldenstrom macroglobulinaemia. 1 Byrd JC Brown JR O'Brien S et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014; 371: 213-223 Crossref PubMed Scopus (1232) Google Scholar , 2 Robak T Burger JA Tedeschi A et al. Single-agent ibrutinib versus chemoimmunotherapy regimens for treatment-naïve patients with chronic lymphocytic leukemia: A cross-trial comparison of phase 3 studies. Am J Hematol. 2018; 93: 1402-1410 Crossref PubMed Scopus (19) Google Scholar , 3 Wang ML Rule S Martin P et al. Targeting BTK with ibrutinib in relapsed or refractory 507 mantle-cell lymphoma. N Engl J Med. 2013; 369: 507-516 Crossref PubMed Scopus (1215) Google Scholar Idelalisib is also an approved B-cell receptor pathway inhibitor. It is a first-in-class, orally-bioavailable, selective competitive inhibitor of phosphoinositol-3-kinase δ (PI3K-δ), which has been approved for the treatment of chronic lymphocytic leukaemia, in association with rituximab, and for follicular lymphoma as monotherapy. 4 Furman RR Sharman JP Coutre SE et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med. 2014; 370: 997-1007 Crossref PubMed Scopus (1369) Google Scholar However, idelalisib can induce severe hepatotoxicity, diarrhoea or colitis, pneumonitis, fatal or serious infections, and intestinal perforation. To address these issues, next-generation PI3K inhibitors, including umbralisib, duvelisib, copanlisib, and buparlisib, are also being investigated for B-cell disorders. These drugs seem to be more potent and better tolerated than idelalisib. Umbralisib is a next-generation inhibitor of PI3K-δ and casein kinase-1ε, and it appears to be well tolerated, has a reduced inhibitory activity on T regulatory cells, and has a lower rate of hepatotoxicity than idelalisib. 5 Burris 3rd, HA Flinn IW Patel MR et al. Umbralisib, a novel PI3Kδ and casein kinase-1ɛ inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study. Lancet Oncol. 2018; 19: 486-496 Summary Full Text Full Text PDF PubMed Scopus (138) Google Scholar Umbralisib also showed a favourable safety profile and good clinical activity when administered as a single drug to patients with B-cell lymphoma in a phase 1 study. 5 Burris 3rd, HA Flinn IW Patel MR et al. Umbralisib, a novel PI3Kδ and casein kinase-1ɛ inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study. Lancet Oncol. 2018; 19: 486-496 Summary Full Text Full Text PDF PubMed Scopus (138) Google Scholar In contrast to idelalisib, patients given umbralisib had no observed cases of grade 3 hepatotoxicity or colitis, and the only grade 3 or higher adverse event was neutropenia. Umbralisib in combination with ibrutinib in patients with relapsed or refractory chronic lymphocytic leukaemia or mantle cell lymphoma: a multicentre phase 1–1b studyUmbralisib plus ibrutinib is well tolerated and active in relapsed or refractory chronic lymphocytic leukaemia and mantle cell lymphoma, with a recommended phase 2 dose of umbralisib 800 mg once daily. To the best of our knowledge, these are the first clinical data on a BTKi and PI3K-δi doublet in B-cell malignancies, and the results suggest that this approach is feasible and worthy of further study. Full-Text PDF

Gene Editing of BTK in Acute Myeloid Leukaemia Using CRISPR-Cas9

Acute myeloid leukemia (AML) blood cancer is found primarily in adults and the elderly, being the third biggest blood cancer killer. In the last decades there has been some improved rates in younger patients, but little improvement in older patients. AML survival rates are the worst of all blood cancers, therefore finding new therapeutic targets to treat AML is crucial.Bruton's tyrosine kinase (BTK) is involved in the signalling of multiple receptors including growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors and integrins. BTK in turn activates many of the major downstream signalling pathways that control cell migration, adhesion, survival and proliferation. BTK is best known for its role in B-cell receptor signalling, but BTK is also expressed and constitutively active in AML cell lines and AML patient blasts. Targeting BTK in B-cell malignancies with the BTK inhibitor ibrutinib has shown clinical effectiveness and tolerability in recent patient trials.Ibrutinib is orally active and covalently binds at the Cys-481 in the ATP-binding domain of BTK, to irreversibly block its enzymatic activity. A proportion of CLL and MCL patients treated with ibrutinib relapse during the therapy, with secondary resistance developing against ibrutinib. This resistance is mainly due to a BTK cysteine to serine mutation at position 481 (C481S) which disrupts ibrutinib's covalent binding to BTK.As little is known about BTK's role and therapeutic potential in AML, we aimed to study the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib. We furthermore generated the BTK-C481S mutation in AML cell lines using the CRISPR-Cas9 gene-editing system. Shown here for the first time is a gene-edited mutation introduced into a human leukemia cell clone that has been purified to homogeneity. We explored the functional impact of BTK-C481S mutation on ibrutinib sensitivity, and mutation-induced adaptation of the phosphoproteome and kinome in AML cells.HEK-293T cells were used for validation purposes to find the most efficient knock-in strategy. We tested the most commonly used CRISPR endonuclease spCas9 as well as two other Cas9 orthologs called SaCas9 and AsCas12a. The designed spacer sequences within the sgRNA that target the gene of interest were delivered either via plasmid or in vitro transcripts. A repair template for homology directed repair (HDR) was designed containing the necessary base changes to change the amino acid at position 481 in BTK from cysteine to serine. The HDR template was delivered via transfection as single-stranded oligodeoxynucleotide (ssODN) or incorporated into plasmid. All strategies tested were successful but due to the poor transfectability of AML cell lines, we used lentiviral delivery to introduce BTK-C481S mutation into AML cell lines.The BTK-C481S mutation was introduced in THP-1 and OCI-AML3 cells. Mutant-positive cells were found via clonal selection and Sanger sequencing. Wild-type (WT) as well as mutant cells were treated with various ibrutinib concentrations ranging from 1 nM to 1 µM and analysed for BTK expression/phosphorylation via Western blot, with effects on cell proliferation, viability and cell cycle measured via flow cytometry. We also performed phosphoproteomics and kinome assays comparing untreated as well as treated (30 nM ibrutinib, 2h) THP-1 WT and THP-1 BTK-C481S cells.Western blot results showed that the effective concentration required for BTK inhibition is 30-fold higher in BTK-C481S mutant compared to WT cells (300 nM vs 10 nM). Wash-out experiments revealed that ibrutinib binding can be reversed in BTK-C481S mutant cells but not WT cells. Little effect of ibrutinib on proliferation and viability were seen comparing BTK-C481S mutant and WT AML cells.These signalling adaptations in BTK-C481S cells are consistent with changes observed in ibrutinib-resistant leukemia patient samples. DisclosuresSlupsky:Verastem: Research Funding.

PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients.Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL).Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line ‘Karpas299‘. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response.Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. DisclosuresParikh:Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Teaching an old pathway new tricks: Targeting BTK to block NLRP3

A proteomics screen identified BTK as a novel inflammasome regulator and demonstrated that blocking BTK can inhibit inflammasome activity.

Targeting BTK through microRNA in chronic lymphocytic leukemia

AbstractBruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Alisertib-Induced Polyploidy and Senescence in DH/DP-DLBCL Is Abrogated By Synthetic Lethal Interactions with Ibrutinib and Rituximab

Diffuse Large B-cell Lymphoma (DLBCL) especially the double-hit (DH)/ double-protein (DP) DLBCL is an aggressive subtype of Non-Hodgkin Lymphoma (NHL). A majority of patients will fail frontline R-CHOP-like therapy and novel agents targeting MYC/BCL2 are needed. Aurora kinases are important regulators of mitosis, frequently over expressed and strongly associated with human cancers including DLBCL. Pre-clinical research and clinical studies have shown that aurora kinase inhibitors have a ~25-30% response rate in B- and T- NHL. Here, we show that alisertib-induced polyploidy and senescence are responsible for treatment failure and can be overcome by implementing a synthetic lethal approach by targeting Bruton's Tyrosine kinase (Btk) with ibrutinib and CD20 with rituximab.It is known that aurora kinases inhibition impede cell cycle progression through mitosis and induce polyploidy in treated cells. However, it is not yet known how these polyploid and senescent cells behave. To further investigate molecular and cellular characteristics of polyploid and senescent cells, we demonstrate these cells are metabolically 4x more active and have fragmented DNA. Though a portion of these cells undergo apoptosis using Caspase pathways, most enter a state of senescence.To assess the effect of alisertib on DH/DP-DLBCL we investigated U2932 and VAL cell lines, where U2932 is a representative of DP-DLBCL that over-expresses MYC, BCL2, BCL6 and mutant TP53, and VAL is representative of DH-DLBCL that carries a three-way-translocation t(8;14;18) and over-expresses BCL2 and MYC. From dose vs. duration experiments, we find 50nM alisertib for 4days of treatment is most effective for investigation of polyploidy and senescent DLBCL cells. We noticed that at day-4, cells appear unusually large in size and 4x more metabolically active compared to controls. This unusually high metabolic rate may have a survival advantage in these cells. To further investigate, we evaluated the DNA integrity within these polyploidy cells. We used the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and found that 98.8% of 8n population had fragmented DNA compared to 19.99% for the 2n population. Then we studied by flow cytometry the fate of the fragmented DNA bearing population using Caspase-3/7 and Annexin-V staining along side with PI and found that only a small fraction of these cells were undergoing apoptosis confirmed by PARP cleavage but the majority of polyploidy cells especially the 8n cells become senescent shown by C12FDG a fluorescent substrate for senescent associated β-galactosidase.We next investigated signaling pathways involved in therapy failure to alisertib treated cells and show that constitutive activation of p-Btk, p-Akt and p-Erk downstream of the BCR signaling pathway. These finding suggest that targeting Btk with ibrutinib and CD20 with rituximab would be a rational therapeutic strategy to partner with alisertib. Cell proliferation MTS assays demonstrated synergistic interactions of alisertib + ibrutinib with low combination Indices (CI) (Chou-Talalay). We next performed alisertib plus ibrutinib + rituximab with a comparison to single agent therapy for polyploidy and senescent abrogation. Triple combination was found to successfully reduce the senescence population (5x lower vs. single agent alisertib), which ultimately results in inducing apoptosis shown by a significant sub-G0 population of cells and increased PARP cleavage. Apoptosis was further confirmed by phosphatidylserine exposure at the cell membrane.Finally, to establish safety and efficacy of the triple combination, we tested a DH/DP-DLBCL SCID mouse U2932 xenograft model. Ibrutinib alone was not active while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ~25%. TGI for Ibrutinib + rituximab was ~50-60%, where the triple combination showed a TGI of >90% (p<0.001). All treatments were well tolerated with stable body weights (<5% body weight loss). Kaplan-Meier survival showed 67% of mice were alive at day 89 in the triple therapy group versus 20% for ibrutinib + rituximab combination, whereas no mice from other treatment group survived passed day 52. Therefore, co-targeting aurora kinase, Btk and CD20 may provide a safe and effective synthetic lethal therapeutic regimen for DH/DP-DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Maintenance therapy in CLL: resolving the controversy

The standard of care for chronic lymphocytic leukaemia is fast changing. For younger patients, frontline chemoimmunotherapy with regimens such as fludarabine, cyclophosphamide, and rituximab 1 Thompson PA Tam CS O'Brien SM et al. Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia. Blood. 2016; 127: 303-309 Crossref PubMed Scopus (381) Google Scholar , 2 Hallek M Fischer K Fingerle-Rowson G et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010; 376: 1164-1174 Summary Full Text Full Text PDF PubMed Scopus (1545) Google Scholar are still appropriate standard-of-care, whereas kinase inhibitors such as ibrutinib are commonly used in older patients, and in those with relapsed disease. 3 Byrd JC Furman RR Coutre SE et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013; 369: 32-42 Crossref PubMed Scopus (1742) Google Scholar Attempts to intensify the regimen of fludarabine, cyclophosphamide, and rituximab with addition of drugs such as mitoxantrone 4 Faderl S Wierda W O'Brien S Ferrajoli A Lerner S Keating MJ Fludarabine, cyclophosphamide, mitoxantrone plus rituximab (FCM-R) in frontline CLL <70 years. Leuk Res. 2010; 34: 284-288 Summary Full Text Full Text PDF PubMed Scopus (39) Google Scholar resulted in additional toxicity and no improvement in therapeutic efficacy. Thus, current efforts are directed towards the use of less toxic drugs such as monoclonal antibodies and lenalidomide as post-induction therapy, commonly referred to as maintenance therapy. Rituximab maintenance versus observation alone in patients with chronic lymphocytic leukaemia who respond to first-line or second-line rituximab-containing chemoimmunotherapy: final results of the AGMT CLL-8a Mabtenance randomised trialRituximab maintenance therapy prolongs progression-free survival in patients achieving at least a PR to induction with rituximab plus chemotherapy, and the treatment is well tolerated overall. Although it is associated with an increase in infections, there is no excess in infection mortality, suggesting that remission maintenance with rituximab is an effective and safe option in the management of chronic lymphocytic leukaemia in early treatment phases. Full-Text PDF

Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies.Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML.Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease.Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253.Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis).We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms.Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients. DisclosuresNo relevant conflicts of interest to declare.

Bruton&amp;rsquo;s tyrosine kinase in chronic inflammation: from pathophysiology to therapy

Bruton's tyrosine kinase (Btk) not only plays a role in differentiation and activa- tion of B-cells but is also involved in regulating signaling in myeloid cell populations, mast cells, platelets, and osteoclasts. Btk plays a critical role in B-cell receptor signaling and has been shown to be involved in CD40 ligation, Toll-like receptor triggering, and Fc-receptor signaling as well, suggesting that targeting Btk might be particularly useful in autoimmune diseases characterized by pathologic antibodies, macrophage activation, and myeloid-derived type I interferon responses. Btk knockout and X-linked immunodeficiency mouse material and X-linked agammaglobulinemia patient samples are powerful tools to examine the role of Btk in health and disease. In addition, the recent development of several covalent and noncovalent small-molecule inhibitors specifically targeting Btk can contribute to our understanding of the functional role of Btk in several cell types and disease mechanisms. Specific Btk inhibitors have been used in both in vitro and in vivo models for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, and Sjogren's syndrome. The current small-molecule Btk inhibitors have demonstrated potent and selective Btk inhibition in preclinical studies, and some inhibitors have propelled the clinical develop- ment of such molecules toward clinical trials in patients with rheumatoid arthritis. Future studies on the effectiveness of Btk inhibition in other autoimmune diseases could further broaden the understanding of the disease mechanisms as well as lead to new targeted therapies.

13‐ICML Satellite symposia programs (updated on May 9, 2015)

13‐ICML Satellite symposia programs (updated on May 9, 2015)

Dual Inhibition of PIM and PI3-Kinase By Ibl-202 Is Highly Synergistic Compared to Mono-Molecular Inhibition and Represents a Novel Treatment Strategy for Chronic Lymphocytic Leukemia

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. [Display omitted] [Display omitted] DisclosuresO’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.