CX-5461 Enhances the Efficacy of APR-246 via Induction of DNA Damage and Replication Stress in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking targeted therapy. Here, we evaluated the anti-cancer activity of APR-246, a P53 activator, and CX-5461, a RNA polymerase I inhibitor, in the treatment of TNBC cells. We tested the efficacy of individual and combination therapy of CX-5461 and APR-246 in vitro, using a panel of breast cancer cell lines. Using publicly available breast cancer datasets, we found that components of RNA Pol I are predominately upregulated in basal-like breast cancer, compared to other subtypes, and this upregulation is associated with poor overall and relapse-free survival. Notably, we found that the treatment of breast cancer cells lines with CX-5461 significantly hampered cell proliferation and synergistically enhanced the efficacy of APR-246. The combination treatment significantly induced apoptosis that is associated with cleaved PARP and Caspase 3 along with Annexin V positivity. Likewise, we also found that combination treatment significantly induced DNA damage and replication stress in these cells. Our data provide a novel combination strategy by utilizing APR-246 in combination CX-5461 in killing TNBC cells that can be further developed into more effective therapy in TNBC therapeutic armamentarium.

Cep55 overexpression promotes genomic instability and tumorigenesis in mice.

High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.

Mechanisms of Genomic Instability in Breast Cancer.

Breast cancer is the most common cancer among women globally. Genomic instability (GI) refers to the increased tendency to accrue genomic alterations. It drives heterogeneity and is a hallmark of cancer. Genomic integrity is closely guarded by several mechanisms, including DNA damage checkpoints, the DNA repair machinery, and the mitotic checkpoint. Alterations in these surveillance mechanisms cause GI. In breast cancer, several pathways maintaining genomic integrity are distinctly altered, including some that have been successfully exploited for therapeutic targeting. In this review, we comprehensively discuss the recent advances on the mechanisms of GI in breast cancer, highlighting DNA repair defects and chromosome segregation errors during mitosis. We further review the clinical implications and therapeutic potential of targeting GI in the era of precision medicine.

MYB regulates the DNA damage response and components of the homology-directed repair pathway in human estrogen receptor-positive breast cancer cells.

Over 70% of human breast cancers are estrogen receptor-positive (ER+), most of which express MYB. In these and other cell types, the MYB transcription factor regulates the expression of many genes involved in cell proliferation, differentiation, tumorigenesis, and apoptosis. So far, no clear link has been established between MYB and the DNA damage response in breast cancer. Here, we found that silencing MYB in the ER+ breast cancer cell line MCF-7 led to increased DNA damage accumulation, as marked by increased γ-H2AX foci following induction of double-stranded breaks. We further found that this was likely mediated by decreased homologous recombination-mediated repair (HRR), since silencing MYB impaired the formation of RAD51 foci in response to DNA damage. Moreover, cells depleted for MYB exhibited reduced expression of several key genes involved in HRR including BRCA1, PALB2, and TOPBP1. Taken together, these data imply that MYB and its targets play an important role in the response of ER+ breast cancer cells to DNA damage, and suggest that induction of DNA damage along with inhibition of MYB activity could offer therapeutic benefits for ER+ breast cancer and possibly other cancer types.

Blockade of PDGFRß circumvents resistance to MEK-JAK inhibition via intratumoral CD8+ T-cells infiltration in triple-negative breast cancer.

BACKGROUND: Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRß in response to JAK2 inhibition. METHODS: Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. RESULTS: We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRß upregulation. Using publically available databases, we showed that patients expressing high PDGFRß or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRß. Thus, co-blockade of PDGFRß with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24- stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. CONCLUSION: These findings reveal a novel regulatory role of JAK2-mediated PDGFRß proteolysis and provide an example of a PDGFRß-mediated resistance mechanism upon specific target inhibition in TNBC.

CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer.

The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers.

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X's role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.

Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells.

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Serological evidence for exposure of dogs to Rickettsia conorii, Rickettsia typhi, and Orientia tsutsugamushi in Sri Lanka.

Vector-borne rickettsial infection is a major cause of febrile illnesses throughout the world. Although vertebrates hosting the vectors play a vital role in the natural cycle of rickettsiae, studies have not been conducted on them in Sri Lanka. Therefore, the present study was designed to determine the exposure of dog population in Rajawatta, Thambavita, and areas of the Western Slopes and Unawatuna of Sri Lanka to rickettsial pathogens. A total of 123 dog blood samples were collected from those areas. Samples were tested for antibodies against Rickettsia conorii (RC) of the spotted fever group (SFG), Rickettsia typhi (RT) of the typhus group (TG), and Orientia tsutsugamushi (OT) of the scrub typhus group (ST) of rickettsiae by indirect immunofluorescence antibody test (IFA). Samples with titers ≥ 1:64 were considered as positive in this study. Collectively, 49% dogs were found to have antibodies against the rickettsial agents. Of the dogs, 42%, 24%, and 2% had antibodies against RC, OT, and RT, respectively. The seropositive rate of 100% was observed in areas of the Western Slopes, whereas the lowest rate of 20% was in Unawatuna. Among the positive samples, antibody titers against RC and OT ranged from 1/64 to 1/8192. In contrast, the few dogs that tested positive for RT showed very low titers of 1/64 and 1/128. Results of this study show the extent of exposure to the pathogen and its dispersion in the natural ecology. We suggest that dogs could be acting as reservoirs in the rickettsial transmission cycle or could be effective tracer animals that can be used to detect areas with potential for future outbreaks.