CXCR4(WHIM) somatic mutations are common Waldenstrom's Macroglobulinemia (WM), and are associated with clinical resistance to ibrutinib. We engineered WM cells to express the most common WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis), CXCR(S338X) mutation in WM. Following SDF-1a stimulation, CXCR4(S338X) WM cells exhibited decreased receptor internalization, enhanced and sustained AKT kinase (AKT) and extracellular regulated kinase (ERK) signaling, decreased poly (ADP-ribose) polymerase and caspase 3 cleavage, and decreased Annexin V staining versus CXCR4 wild-type (WT) cells. CXCR4(S338X)-related signaling and survival effects were blocked by the CXCR4 inhibitor AMD3100. SDF-1a-treated CXCR4(S338X) WM cells showed sustained AKT and ERK activation and decreased apoptotic changes versus CXCR4(WT) cells following ibrutinib treatment, findings which were also reversed by AMD3100. AKT or ERK antagonists restored ibrutinib-triggered apoptotic changes in SDF-1a-treated CXCR4(S338X) WM cells demonstrating their role in SDF-1a-mediated ibrutinib resistance. Enhanced bone marrow pAKT staining was also evident in CXCR4(WHIM) versus CXCR4(WT) WM patients, and remained active despite ibrutinib therapy in CXCR4(WHIM) patients. Last, CXCR4(S338X) WM cells showed varying levels of resistance to other WM relevant therapeutics, including bendamustine, fludarabine, bortezomib and idelalisib in the presence of SDF-1a. These studies demonstrate a functional role for CXCR4(WHIM) mutations, and provide a framework for investigation of CXCR4 inhibitors in WM.
Activating MYD88 mutations are present in 95% of Waldenström macroglobulinemia (WM) patients, and trigger NF-κB through BTK and IRAK. The BTK inhibitor ibrutinib is active in MYD88-mutated (MYD88MUT) WM patients, but shows lower activity in MYD88 wild-type (MYD88WT) disease. MYD88WT patients also show shorter overall survival, and increased risk of disease transformation in some series. The genomic basis for these findings remains to be clarified. We performed whole exome and transcriptome sequencing of sorted tumor samples from 18 MYD88WT patients and compared findings with WM patients with MYD88MUT disease. We identified somatic mutations predicted to activate NF-κB (TBL1XR1, PTPN13, MALT1, BCL10, NFKB2, NFKBIB, NFKBIZ, and UDRL1F), impart epigenomic dysregulation (KMT2D, KMT2C, and KDM6A), or impair DNA damage repair (TP53, ATM, and TRRAP). Predicted NF-κB activating mutations were downstream of BTK and IRAK, and many overlapped with somatic mutations found in diffuse large B-cell lymphoma. A distinctive transcriptional profile in MYD88WT WM was identified, although most differentially expressed genes overlapped with MYD88MUT WM consistent with the many clinical and morphological characteristics that are shared by these WM subgroups. Overall survival was adversely affected by mutations in DNA damage response in MYD88WT WM patients. The findings depict genomic and transcriptional events associated with MYD88WT WM and provide mechanistic insights for disease transformation, decreased ibrutinib activity, and novel drug approaches for this population.
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 CXCR4 WT 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 CXCR4 S338X BCWM.1and MWCL-1 cells despite unaltered BCL2 protein levels (Fig 1A, C). ABT-199 augmented PARP and caspase-3 cleavage in CXCR4 S338X and CXCR4 WT 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 CXCR4 S338X following WT and CXCR4 S338X expressing BCWM.1 (A) and MWCL-1 (B) cells were determined in triplicate sample sets in the presence of CXCL12 (50 nM) 6 h following treatment at indicated doses using a caspase-3/7 cleavage assay. Combination Index >1Á0 denotes synergistic interactions. Apoptotic changes following ABT-199 alone, and in combination with ibrutinib or idelalisib in primary WM cells genotyped for MYD88 and CXCR4 mutations. Results from flow cytometric studies of ex vivo treated WM cells (CD19 + gated) from (C) untreated WM patients (n = 4) or (D) WM patients on active ibrutinib treatment (n = 3). Cells were treated with vehicle control (DMSO), ibrutinib (IB), idelalisib (ID) and ABT-199 (ABT), alone and in combination. P = 0Á02 in untreated WM patient cells comparing any ex vivo treatment versus vehicle control; P ≤ 0Á05 for ABT-199 combinations versus any monotherapy in untreated WM patient cells; P = 0Á04 for ABT-199 versus vehicle control for cells taken from patients on active ibrutinib therapy. All patients expressed MYD88 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 CXCR4 S338X cells treated with ABT-199 alone and with ibrutinib or idelalisib.To further delineate the impact of ABT-199 on ibrutinibor idelalisib-related treatment effects, we used a caspase-3/7 cleavage assay to assess for drug synergy. Synergistic interactions (combination index >1Á0) 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 CXCR4 S338X WM cells, reflecting the prote...
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