The prognostic significance of the aberrant extremes of p53 immunophenotypes in breast cancer

Aims: The utility of p53 as a prognostic assay has been elusive. The aims of this study were to describe a novel, reproducible scoring system and assess the relationship between differential p53 immunohistochemistry (IHC) expression patterns, TP53 mutation status and patient outcomes in breast cancer.

Methods and Results: Tissue microarrays were used to study p53 IHC expression patterns: expression was defined as extreme positive (EP), extreme negative (EN), and non-extreme (NE; intermediate patterns). Overall survival (OS) was used to define patient outcome. A representative subgroup (n = 30) showing the various p53 immunophenotypes was analysed for TP53 hotspot mutation status (exons 4-9). Extreme expression of any type occurred in 176 of 288 (61%) cases. As compared with NE expression, EP expression was significantly associated (P = 0.039) with poorer OS. In addition, as compared with NE expression, EN expression was associated (P = 0.059) with poorer OS. Combining cases showing either EP or EN expression better predicted OS than either pattern alone (P = 0.028). This combination immunophenotype was significant in univariate but not multivariate analysis. In subgroup analysis, six substitution exon mutations were detected, all corresponding to extreme IHC phenotypes. Five missense mutations corresponded to EP staining, and the nonsense mutation corresponded to EN staining. No mutations were detected in the NE group.

Conclusions: Patients with extreme p53 IHC expression have a worse OS than those with NE expression. Accounting for EN as well as EP expression improves the prognostic impact. Extreme expression positively correlates with nodal stage and histological grade, and negatively with hormone receptor status. Extreme expression may relate to specific mutational status.

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

Purpose: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed.

Experimental Design: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays.

Results: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP–specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

BRCA1--conductor of the breast stem cell orchestra:the role of BRCA1 in mammary gland development and identification of cell of origin of BRCA1 mutant breast cancer

Breast cancer treatment has been increasingly successful over the last 20 years due in large part to targeted therapies directed against different subtypes. However, basal-like breast cancers still represent a considerable challenge to clinicians and scientists alike since the pathogenesis underlying the disease and the target cell for transformation of this subtype is still undetermined. The considerable similarities between basal-like and BRCA1 mutant breast cancers led to the hypothesis that these cancers arise from transformation of a basal cell within the normal breast epithelium through BRCA1 dysfunction. Recently, however, a number of studies have called this hypothesis into question. This review summarises the initial findings which implicated the basal cell as the cell of origin of BRCA1 related basal-like breast cancers, as well as the more recent data which identifies the luminal progenitor cells as the likely target of transformation. We compare a number of key studies in this area and identify the differences that could explain some of the contradictory findings. In addition, we highlight the role of BRCA1 in breast cell differentiation and lineage determination by reviewing recent findings in the field and our own observations suggesting a role for BRCA1 in stem cell regulation through activation of the p63 and Notch pathways. We hope that through an increased understanding of the BRCA1 role in breast differentiation and the identification of the cell(s) of origin we can improve treatment options for both BRCA1 mutant and basal-like breast cancer subgroups.

Accumulation of the PX domain mutant Frank-ter Haar syndrome protein Tks4 in aggresomes

BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization.

RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells.

CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF-mutant colorectal cancer cell lines.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor Vemurafenib (PLX4032) has shown significant increases in response rates and overall survival, only minor responses to Vemurafenib treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MEK inhibition in BRAFMT CRC. Methods: Paired BRAFMT/WT RKO and VACO432 CRC cells and non-isogenic BRAFMT LIM2405, WiDR, HT-29 and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interactions between MEK1/2 and JAK1/2 or c-MET inhibition were assessed using the MTT cell viability assays and Flow Cytometry. Apoptosis was measured using Western Blotting for PARP, cleaved caspase 3, 8 and 9, and caspase 3/7 and 8 activity assays. Results: Treatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in synergy and significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or trametinib in BRAFMT CRC cells. The RTK c-MET is activated upstream of STAT3 following MEK1/2 inhibition. Inhibition of c-MET and MEK1/2, using pharmacological inhibitors (crizotinib and AZD6244), results in synergy and increased cell death in BRAFMT CRC cells. Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition in vitro. Combinations of JAK/MEKi or MET/MEKi can be a potential novel treatment strategy for poor prognostic BRAFMT advanced CRC patients.

Abstract B64: The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal cancer.

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor PLX4032 has shown significant increases in response rates and overall survival compared to standard Dacarbazine treatment, only minor responses to PLX4032 treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumors has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MAPK inhibition in BRAFMT CRC.

Methods: Paired BRAFMT/WT RKO and VACO432 CRC cell line models and non-isogenic BRAFMT LIM2405, WiDR and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interaction between MEK1/2 and JAK1/2 inhibition was assessed using the MTT cell viability assays and flow cytometry. Apoptosis was measured using Western blotting for PARP, cleaved caspase 3/8 and caspase 8, 3/7 activity assays.

Results: Treatment with MEK1/2 inhibitors AZD6244, GSK1120212, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or GSK1120212 in BRAFMT CRC cells. In addition, combination of MEK1/2 and JAK/STAT3 inhibition resulted in strong synergy with CI values between 0.3 and 0.7 in BRAFMT CRC cells.

Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition. These data provide a strong rationale for further investigation of combination of MEK1/2 and JAK/STAT3 inhibition in BRAFMT in vivo models.

Addiction to Runx1 is partially attenuated by loss of p53 in the Eµ-Myc lymphoma model

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eµ-Myc model of Burkitt's lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eµ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eµ-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.Leukemia advance online publication, 17 June 2016; doi:10.1038/leu.2016.149.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G₁ -S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Transformation to "high grade" neuroendocrine carcinoma as an acquired drug resistance mechanism in EGFR-mutant lung adenocarcinoma.

Several different acquired resistance mechanisms of EGFR mutant lung adenocarcinoma to EGFR-tyrosine kinase inhibitor (TKI) therapy have been described, most recently transformation to small cell lung carcinoma (SCLC). We describe the case of a 46-year-old female with relapsed EGFR exon 19 deletion lung adenocarcinoma treated with erlotinib, and on resistance, cisplatin-pemetrexed. Liver rebiopsy identified an afatinib-resistant combined SCLC and non-small cell carcinoma with neuroendocrine morphology, retaining the EGFR exon 19 deletion. This case highlights acquired EGFR-TKI resistance through transformation to the high-grade neuroendocrine carcinoma spectrum and that that such transformation may not be evident at time of progression on TKI therapy.