Juraj Bodo

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies

Overexpression of anti-apoptotic BCL-2 family members is a hallmark of many lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) that can be targeted with small molecule inhibitors. ABT-199 is a rationally designed BCL-2 homology (BH)-3 mimetic that specifically binds to BCL-2, but not to MCL-1 and BCL-xL. Although the thrombocytopenia that occurs with navitoclax treatment has not been a problem with ABT-199, clinical trials in CLL could benefit by lowering the ABT-199 concentration through targeting other survival pathways. In this study, we investigated the mechanisms of resistance that develops to ABT-199 therapy by generating ABT-199-resistant (ABT199-R) cell lines via chronic exposure of NHL cell lines to ABT-199. Acquired resistance resulted in substantial AKT activation and upregulation of MCL-1 and BCL-xL levels that sequestered BIM. ABT199-R cells exhibited increased MCL-1 stability and failed to activate BAX in response to ABT-199. The ABT-199 acquired and inherent resistant cells were sensitized to treatment with ABT-199 by inhibitors of the PI3K, AKT, and mTOR pathways, NVP-BEZ235 and GS-1101. NVP-BEZ235, a dual inhibitor of p-AKT and mTOR, reduced MCL-1 levels causing BIM release from MCL-1 and BCL-xL, thus leading to cell death by BAX activation. The PI3Kδ inhibitor GS-1101 (idelalisib) downregulated MCL-1 and sensitized ABT199-R cells through AKT-mediated BAX activation. A genetic approach, through siRNA-mediated down-regulation of AKT, MCL-1, and BCL-xL, significantly decreased cell survival, demonstrating the importance of these cell survival factors for ABT-199 resistance. Our findings suggest a novel mechanism that modulates the expression and activity of pro-survival proteins to confer treatment resistance that could be exploited by a rational combination therapeutic regimen that could be effective for treating lymphoid malignancies.

CpG methylation patterns and decitabine treatment response in acute myeloid leukemia cells and normal hematopoietic precursors

The DNA hypomethylating drug decitabine maintains normal hematopoietic stem cell (HSC) self-renewal but induces terminal differentiation in acute myeloid leukemia (AML) cells. The basis for these contrasting cell fates, and for selective CpG hypomethylation by decitabine, is poorly understood. Promoter CpGs, with methylation measured by microarray, were classified by the direction of methylation change with normal myeloid maturation. In AML cells, the methylation pattern at maturation-responsive CpGs suggested at least partial maturation. Consistent with partial maturation, in gene expression analyses, AML cells expressed high levels of the key lineage-specifying factor CEBPA, but relatively low levels of the key late-differentiation driver CEBPE. In methylation analysis by mass spectrometry, CEBPE promoter CpGs that are usually hypomethylated during granulocyte maturation were significantly hypermethylated in AML cells. Decitabine-induced hypomethylation was greatest at these and other promoter CpGs that are usually hypomethylated with myeloid maturation, accompanied by cellular differentiation of AML cells. In contrast, decitabine-treated normal HSCs retained immature morphology, and methylation significantly decreased at CpGs that are less methylated in immature cells. High expression of lineage-specifying factor and aberrant epigenetic repression of some key late-differentiation driver genes distinguishes AML cells from normal HSCs, and could explain the contrasting differentiation and methylation responses to decitabine.

Combination of ibrutinib with ABT-199: synergistic effects on proliferation inhibition and apoptosis in mantle cell lymphoma cells through perturbation of BTK, AKT and BCL2 pathways

Mantle cell lymphoma (MCL) is considered incurable with a shorter survival than indolent lymphomas. Current standard therapeutic regimens have high initial response rates, however, even with high dose therapy, drug resistance is common and recurrence is anticipated (Garraway & Janne, 2012). Adaptive resistance often results in progression to more clinically aggressive disease. Most patients eventually relapse with a median overall survival of 4–6 years. Thus, more effective therapies are needed. Abnormal activation of Bruton tyrosine kinase (BTK) mediated B-cell-receptor (BCR) signalling pathway contributes to the pathogenesis of B-cell malignancies (Davis et al, 2010). Signals mediated by BTK trigger cell survival pathways such as NFjB, RAS/RAF/MEK/ERK and PI3K/AKT. BTK is therefore an attractive target for inhibition of B-cell growth. A clinical trial suggested Ibrutinib is a promising agent in patients with relapsed or refractory MCL (Wang et al, 2013). However, considering that genetic mutations cause resistance to ibrutinib in chronic lymphocytic leukaemia (CLL) patients and altered signalling pathways are common mechanisms of resistance to single agents, Ibrutinib monotherapy is not expected to cure MCL (Zucca & Bertoni, 2013). The BCL2 family proteins play a critical role in regulating apoptosis and lymphoid malignancies frequently have high BCL2 expression. The BH3-only mimetic ABT-199 selectively inactivates BCL2 and is a promising drug for treatment of BCL2-dependent cancers. However, acquired mutations could cause resistance to ABT-199 in lymphoma cells (Fresquet et al, 2014). These observations indicate potential clinical challenges of applying ABT-199 as a single agent. Combination of ABT199 with other agents may be a strategy to overcome acquired resistance. The effectiveness of ABT-199 as a single agent or in combination is largely unexplored in MCL. Given the presumed non-overlapping pathways, we hypothesized that ibrutinib/ABT-199 combination may overcome the resistance observed with single agents alone. To test this, a new MCL cell line CCMCL1 (Zhao et al, 2013) was examined for the response to these agents. Synergistic inhibition of proliferation was confirmed in 22 of 24 tested combinations, based on combination index (CI) values <1, in which 18 combinations had CI values <0 7, suggesting strong synergy (Fig 1A). Synergistic apoptosis induction was observed in all combinations (Fig 1B). The same assay with four additional MCL cell lines, Jeko-1, Mino, JVM2 and Rec-1, showed strong synergistic effects, as evidenced by CI values <0 7 with a majority being <0 2 (Fig 1C, D). Testing with primary cells from two cases of recurrent MCL also displayed robust synergy of apoptosis induction (Figure S1). Although these two cases displayed different degrees of response to single agents, synergy was observed for both. Another noteworthy finding of this study is molecular mechanisms underlying the interaction of ibrutinib/ABT-199 in MCL cells. In CCMCL1 cells, ibrutinib caused dephosphorylation of BTK(Y223). ABT-199 had no detectable effect on this target but there was a larger decrease of p-BTK(Y223) upon cells co-treated with ibrutinib/ABT-199. A similar effect was observed on p-AKT(S473) (Fig 2A). Immunoblotting of four other MCL cell lines confirmed that this combination enhanced dephosphorylation of the above signalling molecules (Figure S2), which was associated with survival/proliferation of malignant B-cells. The impact of ibrutinib and/or ABT-199 on BCL2 family proteins (BCL2, MCL1 and BCL2L1) was more cell-line dependent. In CCMCL1 cells, ibrutinib alone downregulated MCL1. Each single agent had little effect on BCL2 and BCL2L1, but the combination down-regulated both of these proteins (Fig 2B). Co-treatment of other MCL cell lines with ibrutinib/ABT-199 resulted in decrease of at least one BCL2 family protein (Figure S3). Ibrutinib/ABT-199 co-treatment also more effectively triggered reduced mitochondrial membrane potential compared to single agent, and more poly (ADP-ribose) polymerase (PARP) cleavage (Fig 2C, D) suggested caspase activation. In summary, we report the therapeutic potential of ibrutinib/ABT-199 combination in MCL cells. This combination displayed strongly synergistic effects in all tested cell lines and primary cells from recurrent MCL patients. These cell lines represent different types of MCL, including aggressive MYCtranslocated CCMCL1 (X. Zhao, S. Shetty, M. R. Smith, J. Bodo, E. D. Hsi unpublished data). Mechanistically, ibrutinib/ ABT-199 interaction caused synergistic effects in MCL cells through perturbation of p-BTK and p-AKT mediated survival signals and of BCL2 family proteins (Fig 2E). Ibrutinib was developed as a BTK inhibitor to block BCR signalling. To date, few studies described alterations of BCR signalling causing any effects on expression or function of BCL2 family proteins. However, BTK is necessary for BCR-induced phosphorylation of cAMP-response elementbinding protein (CREB) (Blois et al, 2004). In mature B cells,

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T.

The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways

Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)‐induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform‐specific phosphatidylinositide 3‐kinase (PI3K) inhibitor, GS‐1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non‐Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS‐1101. An interaction of the LBH589/GS‐1101 combination was formally examined by using various concentrations of LBH589 and GS‐1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI‐mediated apoptosis in malignant haematopoietic cells, possibly through both AKT‐dependent or AKT‐ independent mechanisms. Moreover, the increase in HDI‐related apoptosis observed in PI3K inhibitor‐treated cells appears to be related to the disruption of the extracellular signal‐regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

Quantitative In Situ Detection of Phosphoproteins in Fixed Tissues Using Quantum Dot Technology

Detection and quantitation of phosphoproteins (PPs) in fixed tissues will become increasingly important as additional inhibitors of protein kinases enter clinical use and new disease entities are defined by molecular changes affecting PP levels. We characterize fixation conditions suitable for accurate PP quantitation that are achievable in a clinical laboratory and illustrate the utility of in situ quantitation of PPs by quantum dot (QD) nano-crystals in two models: (1) a therapeutic model demonstrating effects of a targeted therapeutic (quantitative reduction of phospho-GSK3β) in xenografts treated with enzas-taurin; and (2) a diagnostic model that identifies elevated levels of nuclear phospho-STAT5 in routine bone marrow biopsies from patients with acute myeloid leukemia based on the presence of the activating FLT3-ITD mutation. Finally, we document production of a well-characterized tissue microarray of widely available cell lines as a multilevel calibrator for validating numerous phosphoprotein assays. QD immunofluorescence is an ideal method for in situ quantitation of PPs in fixed samples, providing valuable cell type—specific and subcellular information about pathway activation in primary tissues. (J Histochem Cytochem 57:701–708, 2009)

Phospho-ERKTHR202/Tyr214 is overexpressed in hairy cell leukemia and is a useful diagnostic marker in bone marrow trephine sections

BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL). We hypothesized that detection of phospho-ERK (pERK) in tissue sections may be a useful marker for diagnosis of HCL. pERK/CD20 double immunostaining was performed on 90 formalin-fixed bone marrow trephine samples affected with small B-cell lymphoproliferative disorders, including 28 cases of HCL. pERK staining was observed in all 28 cases of HCL and in 1 of 62 cases of non-HCL B-cell lymphoproliferative disorders. By allele-specific polymerase chain reaction, all 11 cases of HCL with available DNA were positive for BRAF V600E, as was the 1 pERK+ non-HCL case. The remaining 31 non-HCL cases tested were negative for BRAF V600E. The sensitivity and specificity of pERK for diagnosis of HCL was 100% and 98%, respectively. We conclude that the presence of pERK as detected by immunohistochemical staining is a useful surrogate marker for BRAF V600E in the diagnosis of HCL.

Detection of phospho‐STAT5 in mast cells: a reliable phenotypic marker of systemic mast cell disease that reflects constitutive tyrosine kinase activation

Systemic mastocytosis (SM) is characterized by the abnormal proliferation and accumulation of mast cells (MCs). Constitutive activation of kit, a receptor tyrosine kinase (TK), has been associated with all types of SM. Signal transducers and activators of transcription (STATs), such as STAT5, mediate downstream kit signalling. We hypothesized that nuclear phospho‐STAT5 (pSTAT5) in MCs might reflect TK activation and would be a marker of abnormal MCs in SM. Expression of tryptase, CD25, CD2 and pSTAT5 was evaluated by immunohistochemistry (IHC) on archival cases of SM and cutaneous mastocytosis (CM). pSTAT5 was detected in 23/23 of SM and 1/9 of CM MC nuclei. 23/23 SM had CD25 + MCs. Control tissue MCs were negative for pSTAT5. Nuclear pSTAT5 in MCs from SM reflects abnormal TK activation. We propose nuclear pSTAT5 positivity in MCs as an additional minor phenotypic criterion for diagnosis of SM in future World Health Organization classification schemes.

Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL. The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed. The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.

Angioimmunoblastic T-cell lymphomas with the RHOA p.Gly17Val mutation have classic clinical and pathologic features

Angioimmunoblastic T-cell lymphoma (AITL) is a nodal-based mature T-cell lymphoma with distinctive clinical symptomatology and histology. Research into its pathogenesis supports a cellular derivation from follicular helper T cells and overexpression of genes related to B cells, follicular dendritic cells, and vascular growth. Recently, a novel recurring somatic mutation in RHOA, encoding p.Gly17Val, was discovered in nearly 70% of AITLs and in a smaller proportion of peripheral T-cell lymphomas, not otherwise specified (PTCL-NOS). We investigated a series of AITLs to compare RHOA mutated with wild-type case for clinicopathologic differences. Targeted exome and Sanger sequencing was performed on 27 AITLs and 10 PTCL-NOS. The RHOA G17V mutation was identified in 63% of the AITL cases and in none of the PTCL-NOS cases. The median variant allelic frequency was 14%, with a range of 0.4 to 50% in positive cases. RHOA G17V-mutated cases had a significantly higher incidence of splenomegaly and B symptoms at diagnosis, but there was no difference in overall survival between mutated and wild-type subgroups. Cases with the RHOA G17V mutation had a significantly higher mean microvessel density (P<0.01) and expressed a greater number of follicular helper T-cell markers (P<0.05) than wild-type cases. RHOA G17V is present in a significant proportion of angioimmunoblastic lymphomas and is associated with classic pathologic features of AITL. Additional studies are needed to provide a biological or functional link between altered RHOA function and these pathologic features.