Regulation of the pro-apoptotic BH3-only protein BIM

© 2015 Dr. Leona RohrbeckThis thesis is not authorised to be made available in the Baillieu Reading Room until July 2016The pro-apoptotic BH3-only protein BIM is a member of the BCL-2 family, which is critical for initiation of the intrinsic apoptotic pathway in response to several cytotoxic stimuli. Loss of BIM causes SLE-like autoimmune disease due to accumulation of self-reactive lymphocytes, and promotes tumour formation in humans and mice. In addition, BIM is essential for the killing of cancer cells by several conventional anti-cancer drugs and novel designer drugs that inhibit oncogenic kinases. These findings highlight the importance of BIM in maintaining homeostasis of haematopoietic cells and in functioning as a potent tumour suppressor. Because too much or too little BIM can cause severe abnormalities, expression of BIM needs to be tightly controlled. So far, the vast majority of studies on BIM regulation have been conducted in cell lines, using over-expression systems. Thus more work needs to be done to understand the transcriptional and post-transcriptional regulation of BIM expression. The transcription factor FOXO3a has been reported to be crucial for the initiation of BIM-dependent apoptosis of cytokine-starved haematopoietic cells. I analysed BimΔFoxo/ΔFoxo mice, in which the binding sites for FOXO proteins (including FOXO3a) in the Bim promoters were mutated to prevent FOXO proteins from binding to Bim. In these studies I could show that the development of the haematopoietic system was not impaired in the absence of FOXO-mediated regulation of Bim. In addition, I could demonstrate that cytokine-dependent cells, which I generated from the BimΔFoxo/ΔFoxo mice, were normally sensitive to cytokine withdrawal-induced apoptosis. These data reveal that binding of FOXO proteins to their recognition elements in the Bim promoters is dispensable for BIM expression and for BIM-dependent apoptosis of haematopoietic cells. I then turned my attention to post-transcriptional regulation of Bim because the Bim 3’UTR harbours multiple conserved AU-rich elements (ARE) to which RNA-binding proteins (RBPs) might bind. Several studies demonstrated that binding of RBPs, including members of the CCCH ZFP family, to AREs in the 3’UTR of genes regulate mRNA stability and thereby control gene expression. I generated GFP-Bim 3’UTR reporter constructs in which changes in GFP expression served as a read-out for Bim mRNA stability. In doing this, I identified several proteins of the CCCH ZFP family that act as potential post-transcriptional regulators of Bim mRNA. Additionally, I generated preliminary data that reveal a correlation between mRNA expression of identified ZFPs and Bim mRNA levels in different haematopoietic cell lines. As mentioned above, sensitivity of many cancers to anti-cancer therapies depends on the expression of BIM. Melanoma cells that harbour the BRAFV600E activating mutation initially respond well to treatment with the BRAF kinase specific inhibitor PLX4032, however tumours ultimately acquire resistance to the drug. When hepatocyte growth factor (HGF), which can be secreted from the tumour microenvironment or from the melanoma cells themselves binds to the receptor tyrosine kinase cMET, cMET-expressing melanoma cells acquire resistance to PLX4032-induced apoptosis. I studied the underlying molecular mechanisms of PLX4032-induced killing of BRAF mutant melanoma cells and found that the BH3-only proteins PUMA and to a lesser extent BIM are critical. Moreover, I showed that resistance of BRAF mutant melanoma cells to PLX4032, induced by HGF is accompanied by substantial downregulation of BIM and PUMA proteins. Understanding the mechanisms that lead to resistance of BRAF mutant melanoma cells to PLX4032 will help to develop novel strategies to resensitise these cancer cells to this drug

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells

PPAR-γ promotes type 2 immune responses in allergy and nematode infection

A hallmark of immunity to worm infections and many allergies is a strong type 2 immune response. This is characterized by the production of cytokines interleukin-5 (IL-5) and IL-13 by adaptive T helper 2 (TH2) cells and/or type 2 innate lymphoid cells. Peroxisome proliferator activated receptor-γ (PPAR-γ) is typically regarded as an anti-inflammatory factor. We report that TH2 cells express high levels of PPAR-γ in response to the allergen house dust mite and after infection with the parasite Heligmosomoides polygyrus Mice lacking PPAR-γ in T cells failed to effectively differentiate into IL-5- and IL-13-secreting cells and, hence, did not develop TH2 cell-associated pathologies, including goblet cell metaplasia and eosinophilia, in response to allergen challenge. Furthermore, these mice could not mount protective immune responses to nematode infection. In addition, mice lacking PPAR-γ in T cells had greatly reduced frequencies of TH2 cells in visceral adipose tissue. Mechanistically, PPAR-γ appeared to promote the expression of the IL-33 receptor on the surface of TH2 cells. These results pinpoint PPAR-γ as a factor that drives type 2 responses in allergy, worm infection, and visceral adipose tissue

BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma