The RUNX/CBFß Complex in Breast Cancer: A Conundrum of Context.

Dissecting and identifying the major actors and pathways in the genesis, progression and aggressive advancement of breast cancer is challenging, in part because neoplasms arising in this tissue represent distinct diseases and in part because the tumors themselves evolve. This review attempts to illustrate the complexity of this mutational landscape as it pertains to the RUNX genes and their transcription co-factor CBFß. Large-scale genomic studies that characterize genetic alterations across a disease subtype are a useful starting point and as such have identified recurring alterations in CBFB and in the RUNX genes (particularly RUNX1). Intriguingly, the functional output of these mutations is often context dependent with regards to the estrogen receptor (ER) status of the breast cancer. Therefore, such studies need to be integrated with an in-depth understanding of both the normal and corrupted function in mammary cells to begin to tease out how loss or gain of function can alter the cell phenotype and contribute to disease progression. We review how alterations to RUNX/CBFß function contextually ascribe to breast cancer subtypes and discuss how the in vitro analyses and mouse model systems have contributed to our current understanding of these proteins in the pathogenesis of this complex set of diseases.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

Mitochondrial dynamics regulate genome stability via control of caspase-dependent DNA damage.

Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming. Underlying this process, we reveal a key role for mitochondrial dynamics in the regulation of DNA damage and genome instability. The ability of mitochondrial dynamics to regulate oncogenic DNA damage centers upon the control of minority mitochondrial outer membrane permeabilization (MOMP), a process that enables non-lethal caspase activation leading to DNA damage. Mitochondrial fusion suppresses minority MOMP and its associated DNA damage by enabling homogeneous mitochondrial expression of anti-apoptotic BCL-2 proteins. Finally, we find that mitochondrial dysfunction inhibits pro-apoptotic BAX retrotranslocation, causing BAX mitochondrial localization and thereby promoting minority MOMP. Unexpectedly, these data reveal oncogenic effects of mitochondrial dysfunction that are mediated via mitochondrial dynamics and caspase-dependent DNA damage.

MCL-1 is a clinically targetable vulnerability in breast cancer.

Pro-survival members of the BCL-2 family, including MCL-1, are emerging as important proteins during the development and therapeutic response of solid tumors. Notably, high levels of MCL-1 occur in breast cancer, where functional dependency has been demonstrated using cell lines and mouse models. The utility of restoring apoptosis in cancer cells through inhibition of pro-survival BCL-2 proteins has been realized in the clinic, where the first specific inhibitor of BCL-2 is approved for use in leukemia. A variety of MCL-1 inhibitors are now undergoing clinical trials for blood cancer treatment and application of this new class of drugs is also being tested in solid cancers. On-target compounds specific to MCL-1 have demonstrated promising efficacy in preclinical models of breast cancer and show potential to enhance the anti-tumor effect of conventional therapies. Taken together, this makes MCL-1 an extremely attractive target for clinical evaluation in the context of breast cancer.Abbreviations: ADC (antibody-drug conjugate); AML (Acute myeloid leukemia); APAF1 (apoptotic protease activating factor 1); bCAFs (breast cancer associated fibroblasts); BCL-2 (B-cell lymphoma 2); BH (BCL-2 homology); CLL (chronic lymphocytic leukemia); EGF (epidermal growth factor); EMT (epithelial to mesenchymal transition); ER (estrogen receptor); FDA (food and drug administration); GEMM (genetically engineered mouse model); HER2 (human epidermal growth factor 2); IL6 (interleukin 6); IMM (inner mitochondrial membrane); IMS (intermembrane space); MCL-1 (myeloid cell leukemia-1); MOMP (mitochondrial outer membrane permeabilisation); MM (multiple myeloma); PDX (patient-derived xenograft); OMM (outer mitochondrial membrane); PROTAC (proteolysis-targeting chimeras) TNBC (triple negative breast cancer); UPS (ubiquitin mediated proteolysis system).

Apoptotic stress-induced FGF signalling promotes non-cell autonomous resistance to cell death.

Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair.

CITED2 coordinates key hematopoietic regulatory pathways to maintain the HSC pool in both steady-state hematopoiesis and transplantation.

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic differentiation hierarchy and sustain multilineage hematopoiesis. Here, we show that the transcriptional regulator CITED2 is essential for life-long HSC maintenance. While hematopoietic-specific Cited2 deletion has a minor impact on steady-state hematopoiesis, Cited2-deficient HSCs are severely depleted in young mice and fail to expand upon aging. Moreover, although they home normally to the bone marrow, they fail to reconstitute hematopoiesis upon transplantation. Mechanistically, CITED2 is required for expression of key HSC regulators, including GATA2, MCL-1, and PTEN. Hematopoietic-specific expression of anti-apoptotic MCL-1 partially rescues the Cited2-deficient HSC pool and restores their reconstitution potential. To interrogate the Cited2→Pten pathway in HSCs, we generated Cited2;Pten compound heterozygous mice, which had a decreased number of HSCs that failed to reconstitute the HSC compartment. In addition, CITED2 represses multiple pathways whose elevated activity causes HSC exhaustion. Thus, CITED2 promotes pathways necessary for HSC maintenance and suppresses those detrimental to HSC integrity.

Evasion of cell death: A contributory factor in prostate cancer development and treatment resistance.

Cell death is a natural process in organismal development, homeostasis and response to disease or infection that eliminates unnecessary or potentially dangerous cells and acts as an innate barrier to oncogenesis. Inactivation of cell death is a key step in tumour development and also impedes effective response to cancer therapy. Precise execution of unwanted cells is achieved through regulated cell death processes including the intrinsic apoptotic pathway that is governed by the BCL-2 (B-cell lymphoma 2) protein family. There is compelling evidence that intrinsic apoptosis is defective in prostate cancer, particularly in metastatic and castration resistant advanced disease, currently a lethal diagnosis. New therapeutics have been developed to target pro-survival BCL-2 proteins (including BCL-2, BCL-XL and MCL-1) and show promise in reinstating apoptosis to destroy tumour cells in haematological cancers. Here we discuss perturbation of cell death in prostate cancer and how new therapeutics could improve treatment outcome in prostate cancer.

Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function.

High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1's function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment.

RUNX1 Is a Driver of Renal Cell Carcinoma Correlating with Clinical Outcome.

The recurring association of specific genetic lesions with particular types of cancer is a fascinating and largely unexplained area of cancer biology. This is particularly true of clear cell renal cell carcinoma (ccRCC) where, although key mutations such as loss of VHL is an almost ubiquitous finding, there remains a conspicuous lack of targetable genetic drivers. In this study, we have identified a previously unknown protumorigenic role for the RUNX genes in this disease setting. Analysis of patient tumor biopsies together with loss-of-function studies in preclinical models established the importance of RUNX1 and RUNX2 in ccRCC. Patients with high RUNX1 (and RUNX2) expression exhibited significantly poorer clinical survival compared with patients with low expression. This was functionally relevant, as deletion of RUNX1 in ccRCC cell lines reduced tumor cell growth and viability in vitro and in vivo. Transcriptional profiling of RUNX1-CRISPR-deleted cells revealed a gene signature dominated by extracellular matrix remodeling, notably affecting STMN3, SERPINH1, and EPHRIN signaling. Finally, RUNX1 deletion in a genetic mouse model of kidney cancer improved overall survival and reduced tumor cell proliferation. In summary, these data attest to the validity of targeting a RUNX1-transcriptional program in ccRCC. SIGNIFICANCE: These data reveal a novel unexplored oncogenic role for RUNX genes in kidney cancer and indicate that targeting the effects of RUNX transcriptional activity could be relevant for clinical intervention in ccRCC.

Correction: BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas.

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.

Targeting BCL-2 regulated apoptosis in cancer.

The ability of a cell to undergo mitochondrial apoptosis is governed by pro- and anti-apoptotic members of the BCL-2 protein family. The equilibrium of pro- versus anti-apoptotic BCL-2 proteins ensures appropriate regulation of programmed cell death during development and maintains organismal health. When unbalanced, the BCL-2 family can act as a barrier to apoptosis and facilitate tumour development and resistance to cancer therapy. Here we discuss the BCL-2 family, their deregulation in cancer and recent pharmaceutical developments to target specific members of this family as cancer therapy.

MCL-1 is a prognostic indicator and drug target in breast cancer.

Analysis of publicly available genomic and gene expression data demonstrates that MCL1 expression is frequently elevated in breast cancer. Distinct from other pro-survival Bcl-2 family members, the short half-life of MCL-1 protein led us to investigate MCL-1 protein expression in a breast cancer tissue microarray and correlate this with clinical data. Here, we report associations between high MCL-1 and poor prognosis in specific subtypes of breast cancer including triple-negative breast cancer, an aggressive form that lacks targeted treatment options. Deletion of MCL-1 in the mammary epithelium of genetically engineered mice revealed an absolute requirement for MCL-1 in breast tumorigenesis. The clinical applicability of these findings was tested through a combination of approaches including knock-down or inhibition of MCL-1 to show triple-negative breast cancer cell line dependence on MCL-1 in vitro and in vivo. Our data demonstrate that high MCL-1 protein expression is associated with poor outcome in breast cancer and support the therapeutic targeting of MCL-1 in this disease.

Corrigendum: Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

This corrects the article DOI: 10.1038/nature22056.

Mnt modulates Myc-driven lymphomagenesis.

The transcriptional represser Mnt is a functional antagonist of the proto-oncoprotein Myc. Both Mnt and Myc utilise Max as an obligate partner for DNA binding, and Myc/Max and Mnt/Max complexes compete for occupancy at E-box DNA sequences in promoter regions. We have previously shown in transgenic mouse models that the phenotype and kinetics of onset of haemopoietic tumours varies with the level of Myc expression. We reasoned that a decrease in the level of Mnt would increase the functional level of Myc and accelerate Myc-driven tumorigenesis. We tested the impact of reduced Mnt in three models of myc transgenic mice and in p53+/- mice. To our surprise, mnt heterozygosity actually slowed Myc-driven tumorigenesis in vavP-MYC10 and Eµ-myc mice, suggesting that Mnt facilitates Myc-driven oncogenesis. To explore the underlying cause of the delay in tumour development, we enumerated Myc-driven cell populations in healthy young vavP-MYC10 and Eµ-myc mice, expecting that the reduced rate of leukaemogenesis in mnt heterozygous mice would be reflected in a reduced number of preleukaemic cells, due to increased apoptosis or reduced proliferation or both. However, no differences were apparent. Furthermore, when mnt+/+ and mnt+/- pre-B cells from healthy young Eµ-myc mice were compared in vitro, no differences were seen in their sensitivity to apoptosis or in cell size or cell cycling. Moreover, the frequencies of apoptotic, senescent and proliferating cells were comparable in vivo in mnt+/- and mnt+/+ Eµ-myc lymphomas. Thus, although mnt heterozygosity clearly slowed lymphomagenesis in vavP-MYC10 and Eµ-myc mice, the change(s) in cellular properties responsible for this effect remain to be identified.

Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.