The <scp>BCL</scp>2 antagonist <scp>ABT</scp>‐199 triggers apoptosis, and augments ibrutinib and idelalisib mediated cytotoxicity in <i><scp>CXCR</scp>4</i><sup><i>Wild‐type</i></sup> and <i><scp>CXCR</scp>4</i><scp><sup><i>WHIM</i></sup></scp> mutated Waldenstrom macroglobulinaemia cells (2015)

Whole genome sequencing has revealed MYD88L265P and CXCR4WHIM somatic mutations in Waldenstrom's Macroglobulinemia (WM) (Treon et al, 2012; Hunter et al, 2014). MYD88L265P supports WM cell survival through divergent pathways including Bruton Tyrosine Kinase (BTK) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PI3KCD, also termed PI3Kδ) (Yang et al, 2013a,b). Ibrutinib targets BTK, while idelalisib targets PI3KCD. Ibrutinib and idelalisib are active in previously treated WM patients, with overall response rates of 87% and 70%, respectively (Treon et al, 2013; Gopal et al, 2014). Most responses are partial and no complete responses have been observed. CXCR4WHIM mutations confer in vitro ibrutinib and idelalisib resistance (Cao et al, 2014; Roccaro et al, 2014) and CXCR4WHIM-mutated WM patients have lower overall and major responses to ibrutinib (Treon et al, 2013). AKT1 and MAPK1 are hyper-activated in CXCR4WHIM-engineered WM cells following CXCL12 stimulation, and AKT1 is constitutively activated in WM cells from CXCR4WHIM patients on ibrutinib (Cao et al, 2014). The hyper-activation of AKT1 and MAPK1 may contribute to ibrutinib resistance in CXCR4WHIM-mutated WM patients (Cao et al, 2014). BCL2 is an anti-apoptotic protein that confers resistance to many anti-neoplastic agents used in B-cell malignancies. BCL2 is overexpressed in primary WM cells (Chng et al, 2006). ABT-199 is a highly selective BCL2 antagonist that is clinically active in B-cell malignancies, with major responses observed in 3 of 4 previously treated WM patients (Davids et al, 2014). These findings suggest that BCL2 contributes to WM cell survival. We therefore investigated whether BCL2 also protects against ibrutinib- and idelalisib-related apoptosis in CXCR4WT and CXCR4WHIM WM cells. Written consent was obtained for use of patient samples per our Institutional Review Board. Development of CXCR4WT and CXCR4S338X BCWM.1 and MWCL-1 cells was previously reported (Cao et al, 2014). Ibrutinib and idelalisib were obtained from MedChem Express (Monmouth Junction, NJ, USA), and ABT-199 from Selleck Chemicals (Houston, TX, USA). BCL2 levels were determined by immunoblotting (Abcam, Cambridge, MA, USA). For survival assessment, WM cells underwent immunoblotting for cleaved PARP and caspase-3 (Abcam), and Annexin V staining (R&D Systems, Minneapolis, MN, USA) by flow cytometry. Synergistic drug interactions were determined by the Caspase-Glo® 3/7 assay (Promega, Madison WI, USA) in the presence of CXCL12 (50 nmol/l). CXCR4WT and CXCR4S338X BCWM.1 and MWCL-1 cells expressed BCL2 protein, with higher levels observed in the latter. BCL2 levels were not impacted by CXCR4 mutation status, or CXCL12 stimulation (Fig 1A). Treatment of CXCR4WT and CXCR4S338X BCWM.1 and MWCL-1 cells with ibrutinib or idelalisib induced caspase-3 and PARP cleavage at 6 h (Fig 1B), though modest Annexin V staining, denoting full apoptotic progression, was observed at 18 and 40 h (data not shown). CXCL12 abrogated ibrutinib- or idelalisib-related PARP and/or caspase-3 cleavage, and was more pronounced in CXCR4S338X WM cells. Similarly, ABT-199 induced PARP and caspase-3 cleavage in CXCR4S338X and CXCR4WT BCWM.1 and MWCL-1 cells. PARP and caspase-3 cleavage was less pronounced in MWCL-1 cells regardless of CXCR4 mutation status, and probably reflected higher BCL2 protein levels (Fig 1A). CXCL12 also abrogated PARP and caspase-3 cleavage in ABT-199 treated CXCR4S338X BCWM.1 and MWCL-1 cells despite unaltered BCL2 protein levels (Fig 1A, C). ABT-199 augmented PARP and caspase-3 cleavage in CXCR4S338X and CXCR4WT BCWM.1 and MWCL-1 WM cells treated with ibrutinib or idelalisib (Fig 1B), and led to increased Annexin V staining (Fig 1D). CXCL12 abrogated PARP and/or caspase-3 cleavage (Fig 1B), and Annexin V staining (data not shown) more so in CXCR4S338X following ABT-199 treatment with either ibrutinib or idelalisib. Addition of the CXCR4 antagonist AMD3100 abrogated CXCL12 protective effects, and augmented PARP and caspase-3 cleavage (Fig 1B), and Annexin V staining (data not shown) in CXCR4S338X cells treated with ABT-199 alone and with ibrutinib or idelalisib. To further delineate the impact of ABT-199 on ibrutinib- or idelalisib-related treatment effects, we used a caspase-3/7 cleavage assay to assess for drug synergy. Synergistic interactions (combination index >1·0) were evident at nearly all dose combinations of ABT-199 with either ibrutinib or idelalisib in CXCR4WT BCWM.1 and MWCL-1 cells in the presence of CXCL12. Synergistic interactions were also evident, though at higher dose combinations of ABT-199, with either ibrutinib or idelalisib in CXCR4S338X WM cells, reflecting the protective effects of CXCL12 in CXCR4WHIM-mutated cells (Fig 2A, B). The impact of ABT-199 on ibrutinib- or idelalisib-related treatment effect was also assessed in bone marrow (CD19-sorted) WM cells from 4 (2 MYD88L265PCXCR4WT; 2 MYD88L265PCXCR4S338X) untreated WM patients within their microenvironment. Ex vivo treatment of these cells with ABT-199 resulted in both direct (P = 0·02) and enhanced apoptosis when combined with either ibrutinib or idelalisib (P ≤ 0·05 vs. any monotherapy) regardless of CXCR4 mutation status (Fig 2A). We also examined ABT-199 in primary WM cells within their microenvironment from three patients (1 MYD88L265PCXCR4WT; 2 MYD88L265PCXCR4S338X) on active ibrutinib treatment for ≥6 months (Fig 2C). While ex vivo ibrutinib treatment had little effect, treatment with ABT-199 induced apoptosis in WM cells from two of three patients (Fig 2D). In these studies, we sought to clarify if BCL2 expression was impacted by CXCR4WHIM mutations, and whether BCL2 contributed to ibrutinib and idelalisib resistance in CXCR4WHIM-mutated WM cells. Despite the lack of BCL2 modulation by CXCL12, BCL2 protected against ibrutinib- and idelalisib-triggered apoptosis in CXCR4WT and CXCR4S338X BCWM.1 and MWCL-1 cells. ABT-199 triggered apoptosis in WM cells regardless of CXCR4WHIM mutation status and prior ibrutinib exposure. However, more robust CXCL12 protection against ABT-199 was observed in CXCR4S338X-mutated cells, a finding that could be related to AKT1 and MAPK1 hyper-activation (Cao et al, 2014). AMD3100 blocked the protective effects of CXCL12 in WM cells treated with ABT-199 alone, and with ibrutinib or idelalisib. The long-term safety and efficacy of AMD3100 administration was recently demonstrated in WHIM-syndrome patients, and provides a paradigm for CXCR4 inhibition in WM patients. Our findings therefore show that ABT-199 induces direct apoptosis, and enhances ibrutinib and idelalisib triggered apoptosis in both CXCR4WT and CXCR4WHIM WM cells. These findings may be relevant to other therapeutics, and support further investigation of ABT-199 alone, and in combination strategies in WM patients. The authors acknowledge the generous support of the Leukemia and Lymphoma Society for a Translational Research Grant, Peter S. Bing M.D., the Edward and Linda Nelson Fund for WM Research, the Kerry Robertson Fund for WM Studies, in support of these studies. YC, GY and SPT conceived and designed the experiments. SPT, GY, ZRH performed the data analysis. JC, XL, LX, YC, GY, EH, and NT performed the laboratory experiments. SK, JJC, MSD, and SPT participated in patient care, sample and data collection. SPT wrote the manuscript. SPT received research funding, speaking honoraria and/or consulting fees from Pharmacyclics Inc., Janssen Pharmaceuticals Inc., and Gilead Pharmaceuticals, Inc. MSD received research funding from Pharmacyclics, Inc., consulting fees from Janssen Pharmaceuticals, Inc., Gilead Pharmaceuticals, Genentech, and Infinity Pharmaceuticals.