Matthias Versele

The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation

The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their “dependence” on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.

Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy

Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy.

Surviving Metabolic Stress: Of Mice (Squirrels) and Men

Understanding how cancer cells survive harsh environmental conditions may be fundamental to eradicating malignancies proven to be impervious to treatment. Nutrient and growth factor deprivation, hypoxia, and low pH create metabolic demands that require cellular adaptations to sustain energy levels. Protein synthesis is one of the most notable consumers of energy. Mounting evidence implicates exquisite control of protein synthesis as a survival mechanism for both normal and malignant cells. In this commentary, we discuss the role of protein synthesis in energy conservation in cancer and focus on elongation factor-2 kinase, a downstream component of the PI3K-AKT pathway that behaves as a critical checkpoint in energy consumption. .

Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)–mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246–56. ©2017 AACR.

Identification of potential ibrutinib combinations in hematological malignancies using a combination high-throughput screen

Abstract Matrix high-throughput screening (HTS) methods are increasingly employed to rapidly define potential therapeutic drug combinations. We used combination HTS to identify compounds showing synergistic anti-proliferative activity with ibrutinib, an irreversible, small-molecule inhibitor of Bruton’s tyrosine kinase. The goal was to identify ibrutinib combinations with maximum synergistic effects in heme malignancy lines, particularly in non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). Growth inhibition (GI) was used to measure cell viability; synergy scores characterized strength of synergistic interaction. Single-agent ibrutinib demonstrated varying degrees of activity across 30 cell lines evaluated. In DLBCL lines, TMD8 was the most sensitive to ibrutinib (GI50 = 0.001); combinations with BCL-2 inhibitor ABT-199, and PI3K inhibitors IPI-145 and GDC-0941 showed the strongest synergistic activity. Anti-proliferative synergies were also observed with BET bromodomain inhibitor (+)-JQ1, XPO1 inhibitor selinexor, and IRAK4 inhibitor, and confirmed using apoptosis assay. These findings are intended to inform and advance treatment of B-cell malignancies.

Abstract 3229: Managing stress: Discovery of inhibitors of the atypical kinase eEF2K and the class III PI3K, VPS34

Adaptation to nutrient deprivation in the tumour microenvironment was recently shown to be dependent on the appropriate regulation of protein elongation rate through activation of the atypical kinase, eukaryotic elongation factor 2 kinase (eEF2K) (Leprivier et al., 2013, Cell 153(5):1064-79). We have solved the crystal structure of the kinase domain of eEF2K, and used structure-based design as well as screening approaches to optimize a chemical series into single-digit nM inhibitors of eEF2K, with remarkable selectivity across the protein kinome (only 5-10 kinases out of 400 tested are inhibited to more than 50% at 1 μM). These compounds inhibit the phosphorylation of eEF2 in nutrient-starved or metabolically stressed cells, and increase protein elongation rates through stabilization of the ribosomal elongation complex under stress. Evotec9s Cellular Target Profiling of these compounds in cell lysates, revealed that a subset of the eEF2K inhibitors also bind with low nM affinity to the class III phosphatidylinositol-3-kinase, VPS34, but not to class I or II PI3Ks, and pull down the entire beclin-UVRAG-VPS34 complex. Proteomic and biochemical screening of the compound set enabled deconvolution of potent EF2K versus VPS34 inhibitors. Inhibition of VPS34 results in abrogation of autophagic flux, as indicated by rapid and massive accumulation of p62, and impairs survival in specific subsets of tumor cell lines, consistent with a pro-survival role for autophagy in those models (Cheng et al., 2013, Pharmacol Rev 65(4):1162-97). Interestingly, a whole-genome pooled shRNA screen in a KRAS/PI3KCA mutant colorectal cancer cell line revealed that reduction of beclin levels significantly increased sensitivity to VPS34 inhibition. In contrast, inhibition of eEF2K does not appear to be anti-proliferative across a wide panel of cancer cell lines under standard cell culture conditions. Our work has provided the first potent inhibitors to unravel the functional relevance of eEF2K and VPS34 in adaptation to cellular stress, and to examine the utility of inhibiting these kinases in nutrient-deprived and/or autophagy-addicted tumours. Citation Format: Matthias Versele, Claire Moore, Christopher G. Proud, Cindy Rockx, Inez Van de Weyer, Kurt Van Baelen, Stephanie Blencke, Sebastian K. Wanndinger, Gaston Diels, Didier Berthelot, Marcel Viellevoye, Bruno Schoentjes, Berthold Wroblowski, Lieven Meerpoel, William N. Hait. Managing stress: Discovery of inhibitors of the atypical kinase eEF2K and the class III PI3K, VPS34. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3229. doi:10.1158/1538-7445.AM2014-3229

The effect of Bruton's tyrosine kinase (BTK) inhibitors on collagen-induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular carcinoma (TEC)

Brutons tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) are expressed by human platelets. These kinases participate in platelet activation through the collagen receptor glycoprotein VI and may perform overlapping functions. In clinical studies, BTK inhibitors (ibrutinib, acalabrutinib, tirabrutinib, zanubrutinib) have been associated with increased bleeding risk, which may result from inhibition of BTK alone or of both BTK and TEC, although the role of TEC in bleeding risk remains unclear.

Abstract 365: High throughput screen to evaluate combinations with ibrutinib in various B-cell malignancies

The primary objective of this study was to discover synergies leading to mechanistic insights and novel combinations for ibrutinib, a small-molecule inhibitor of Bruton9s tyrosine kinase (BTK). Ibrutinib has been approved for relapsed/refractory (R/R) and del(17p) chronic lymphocytic leukemia, and R/R mantle-cell lymphoma (MCL). BTK is part of the B-cell receptor (BCR) signaling pathway, so it is of interest to examine synergy between ibrutinib and agents that target other aspects of the BCR pathway. These include PI3K and IRAK inhibitors and apoptosis inhibitors. Combinations were evaluated in a high-throughput, tumor microenvironment-directed format. Ibrutinib was combined with inhibitors of the BCR pathway and apoptosis, whose targets included MCL-1, BCL2, XPO1, and isoforms of PI3K, IRAK, and BRD. Histologies examined included follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), MCL, acute myeloid leukemia (AML), acute B-lymphoblastic leukemia, and Burkitt9s lymphoma. Cell lines were screened in the presence of human marrow stromal-cell-conditioned media and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies in a 72-hour cell viability ATP lite assay (384-well plate, 9×9 optimized matrix, 4 replicates). Dose response matrix screening was used to measure combination effects, which manifest as potency shifts or efficacy boosts. Combination effects can be characterized by comparing each data point to a combination reference model derived from single-agent curves using the Loewe additivity model. Ibrutinib demonstrated varying activity across cell lines. Combination activity was classified based on synergy score raw values. The best combination among tested compounds was with ABT-199, a BCL2i. This combination showed high or medium synergy in 2/5 DLBCL, 3/4 FL, and 2/5 MCL cell lines; this was also the only agent to show good synergy in AML (3/5 lines). Interestingly, ABT-737, a BCL2i that also targets BCL-XL, has previously been shown to be synergistic with ibrutinib in DLBCL lines. Synergy was also seen with other apoptotic agents such as MCL-1i and the epigenetic BETi JQ-1 across B-NHL, but in fewer lines. Among the PI3Ki, PI3Kα/δi did not show much activity, but the PI3Kδ/γi IPI-145 was synergistic in 2/5 DLBCL, 3/4 FL cell lines, and 1/5 MCL cell lines, a pattern very similar to the PI3Kδi CAL-101 (idelalisib). Ibrutinib also combined well with IRAK1/4i and XPO1i selinexor, with high activity in 1/5 DLBCL cell lines each, and medium activity in 1/5 DLBCL, 1/4 FL, and 2/5 MCL (XPO1i) and 1/5 DLBCL, 2/4 FL, and 1/5 MCL cell lines (IRAK1/4i). Synergy was mostly observed in B-cell malignancies, but interesting synergy was observed with ABT-199 in AML. In B-NHL, ibrutinib combined with BCL2i and PI3Ki showed the best combination activity. Ibrutinib was also synergistic with other agents in selected B-NHL lines with no observed antagonism, suggesting that further study in specific histologies is warranted. Citation Format: Shalini Chaturvedi, Michael Schaffer, Cuc Davis, Regina Aquino, Emily Stepanchick, Matthias Versele, Sriram Balasubramanian. High throughput screen to evaluate combinations with ibrutinib in various B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 365.

Abstract 4800: Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia

Inhibition of Flt3 kinase activity is a promising strategy for the treatment of Flt3 mutant acute myeloid leukemia (AML). However, clinical studies with Flt3 kinase inhibitors have shown that mutations of the gate keeper or an activation loop residue in the Flt3 catalytic domain limits the duration of response. Such mutations generally reduce kinase domain binding affinity and residence time of the kinase inhibitor, and consequently its efficacy. We therefore investigated whether covalent, irreversible binding to Flt3 could overcome some of the limitations of classic non-covalent Flt3 inhibitors. Here, we report for the first time on the high-affinity Cys828-covalent binding mode of a resorcylic acid lactone to the isolated Flt3 kinase domain using X-ray crystallography and kinetic binding assays. In a cellular context (Ba/F3-Flt3-ITD), mutation of Cys828 to Ala reduces potency (IC50) from low nM to microM, demonstrating all relevant Flt3 inhibition in cells critically depends on Cys828. Consistently, the molecule is a low nM inhibitor of both Flt3-ITD and Flt3D835Y in vitro and in BaF3 cells, translating to potent nM inhibition of Flt3-ITD driven AML cell line proliferation, with only microM antiproliferative activity in non-Flt3 driven AML and unrelated leukemia cell lines. Finally, in spite of its fast clearance when dosed to MV4-11 (Flt3-ITD AML) xenograft bearing mice, robust anti-tumor activity was observed using a once-daily treatment schedule. These data demonstrate the feasibility of covalent Flt3 inhibition, and suggest it represents an attractive novel therapeutic approach for the treatment of Flt3-driven AML. Citation Format: Matthias Versele, Burkhard Haefner, Berthold Wroblowski, Ian Stansfield, Laurence Mevellec, Ron Gilissen, Lars Neumann, Martin Augustin, Kris Jacobs, Jan Cools, S Barluenga, M Rothlingshofer, G Karthikeyan, Ricardo Attar, Lieven Meerpoel, Nicolas Winssinger. Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4800.

Abstract 3228: Characterization of a novel, orally bioavailable, potent and highly selective small molecule inhibitor of PERK: A tool to probe the biphasic concentration-dependent induction of ER stress in models of multiple myeloma and B-cell lymphoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stress and the accumulation of malfolded proteins in the endoplasmic reticulum (ER). In highly secretory malignancies, such as multiple myeloma (MM), characteristically there is both an extensively developed ER and a markedly elevated UPR to ensure a homeostatic balance between ER burden and ER capacity - thus making components of the UPR attractive targets for therapeutic intervention. One such target is PERK (PKR-like endoplasmic reticulum kinase), a member of the eukaryotic initiation factor (eIF)2α kinase family, that is activated during the UPR to chronic ER stress. Here, we describe the identification and characterization of compound (1) - a novel, orally bioavailable, potent and highly selective small molecule inhibitor of PERK. (1) is a sub-nM inhibitor of PERK, with a >100-fold window against other kinases (including other eIF2α kinases) - targeting only 6 out of a 400 kinase panel with sub-μM IC50s. It inhibits phosphorylation of eIF2α at 10 nM (IC50) in HEK293 cells (incubated with the ER stressor tunicamycin) and proves to be selectively anti-proliferative in an ER-stressed epithelial cancer model (A549 cells with tunicamycin) at nM concentrations, but to a lesser extent in the absence of ER stress. Furthermore, in the absence of an exogenous ER stressor, (1) induced excessive ER stress (eg, as evidenced by induction of the pro-apoptotic CHOP gene), and decreased cell viability selectively in MM cell lines and certain B-cell lymphoma lines at low nM concentrations, but not in normal or malignant epithelial cells. However, despite a sustained impact on eIF2α phosphorylation, interestingly compound (1) elicited a biphasic concentration dependent induction of ER stress (and consequent inhibition of cell viability) - with ER stress induction maximal at inhibitor concentrations corresponding to circa 50-75 % inhibition of PERK, returning progressively to baseline with concentrations rising to > IC90. Whole-genome expression profiling revealed that all significant changes seen at 10-100 nM of (1), returned to baseline levels at 1000 nM, suggesting a compensatory mechanism very proximal to phosphorylation of eIF2α. In summary, the highly selective PERK inhibitor, (1), validated the unique sensitivity of subsets of multiple myeloma and B-cell lymphoma to disruption of the UPR, but unexpectedly revealed a compensatory mechanism associated with potent PERK inhibition. Citation Format: Ian Stansfield, Yannick Ligny, Yvan Simonnet, Christophe Demestre, Nathalie Amblard, Christophe Meyer, Tamara Geerts, Jeroen Van de Ven, Ilse Van den Wyngaert, Peter Vermeulen, Inge Beerden, Danielle Peeters, Johnny Liebregts, Kurt Van Baelen, Cedric Simillion, Boudewijn Janssen, Tinne Verhulst, Norbert Esser, James Bischoff, Lieven Meerpoel, Matthias Versele. Characterization of a novel, orally bioavailable, potent and highly selective small molecule inhibitor of PERK: A tool to probe the biphasic concentration-dependent induction of ER stress in models of multiple myeloma and B-cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2014-3228