The impressive selectivity and efficacy of BH3 mimetics for treating cancer has largely been limited to BCL-2 dependent hematological malignancies. Most solid tumors depend on other anti-apoptotic proteins, including MCL-1, for survival. The recent description of S63845 as the first specific and potent MCL-1 inhibitor represents an important therapeutic advance, since MCL-1 is not targeted by the currently available BH3 mimetics, Navitoclax or Venetoclax, and is commonly associated with chemoresistance. In this study, we confirm a high binding affinity and selectivity of S63845 to induce apoptosis in MCL-1-dependent cancer cell lines. Furthermore, S63845 synergizes with other BH3 mimetics to induce apoptosis in cell lines derived from both hematological and solid tumors. Although the anti-apoptotic BCL-2 family members in these cell lines interact with a spectrum of pro-apoptotic BH3-only proteins to regulate apoptosis, these interactions alone do not explain the relative sensitivities of these cell lines to BH3 mimetic-induced apoptosis. These findings necessitated further investigation into the requirement of BH3-only proteins in BH3 mimetic-mediated apoptosis. Concurrent inhibition of BCL-X and MCL-1 by BH3 mimetics in colorectal HCT116 cells induced apoptosis in a BAX- but not BAK-dependent manner. Remarkably this apoptosis was independent of all known BH3-only proteins. Although BH3-only proteins were required for apoptosis induced as a result of BCL-X inhibition, this requirement was overcome when both BCL-X and MCL-1 were inhibited, implicating distinct mechanisms by which different anti-apoptotic BCL-2 family members may regulate apoptosis in cancer.

The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XL specific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-X L (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.

U-CLL (Unmutated-Chronic Lymphocytic Leukaemia) and M-CLL (Mutated-Chronic Lymphocytic Leukaemia),suggesting a possible correlation between the IBtk expression levels and aggressiveness of disease. Recently, we provided the evidence of IBtk as a prognostic marker of CLL progression. Material and methods Ibtk -/-mice were bred with congenic Em-myc transgenic mice, a pre-clinical mouse model of NonHodgkin's Lymphoma, to generate Ibtk -/-Em-myc mice, which were monitored for survival and tumour development. We used flow-cytometry for B cells immunophenotyping. Primary murine B cells were compared using different approaches: proliferation, cell viability and cell death assays. Results and discussions Our data provide the evidence that Ibtk gene increases survival and delays tumour onset in Emmyc mice.Ibtk -/-Em-myc mice mostly developed pre-B lymphoma and to a lesser extent mature B lymphoma, which was consistent with the tumour phenotype of Em-myc mice.Loss of Ibtk substantially reduced the number of premalignant Blymphoid cells without affecting their proliferation rate. In particular, pre-cancerous immature B cells were reduced in bone marrow and spleen of Ibtk -/-Em-myc compared to Ibtk +/ + Em myc mice.In Em-myc mice, the pre-cancerous state is characterised by aberrant proliferation of B-lymphoid cells, which is initially offset by pro-apoptotic action of c-Myc.We have previously shown that the enhanced expression of IBtk in CLL cells down regulate the expression of pro-apoptotic genes, thus counteracting apoptosis.According to this hypothesis, we found an increased spontaneous apoptosis of pre-cancerous Ibtk Introduction The ability to resist anoikis, a form of apoptosis triggered by detachment of non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumour invasion and metastasis. ErbB2/Her2, an oncoprotein often overproduced by breast cancer cells, blocks anoikis of breast cancer cells via mechanisms that are not well understood. Our goal is to understand these mechanisms. Material and methods We studied the expression of a transcription factor Irf6, an important mediator of the mammary gland homeostasis, in non-malignant breast epithelial cells MCF-10A and non-malignant primary human mammary epithelial cells (HMEC) before and after their detachment from the ECM. We also examined Irf6 expression in detached ErbB2-overproducing breast cancer cells. We used small interfering RNAs (siRNAs) to knock down Irf6 in the non-malignant cells, infected breast tumour cells with an Irf6-encoding retrovirus to overexpress Irf6 and measured detachmentinduced apoptosis of the cells before and after the indicated changes in Irf6 expression. Results and discussions We found that detachment of MCF-10A or HMEC cells upregulates Irf6 and that Irf6 upregulation promotes their anoikis. We established that ErbB2 downregulates Irf6 in detached breast cancer cells. We also found that an anti-ErbB2 antibody trastuzumab and a small molecule ErbB2 inhibitor lapatinib used for ErbB2-po...