Complexation of histone deacetylase inhibitor belinostat to Cu(II) prevents premature metabolic inactivation in vitro and demonstrates potent anti-cancer activity in vitro and ex vivo in colon cancer.

PURPOSE: The histone deacetylase inhibitor (HDACi), belinostat, has had limited therapeutic impact in solid tumors, such as colon cancer, due to its poor metabolic stability. Here we evaluated a novel belinostat prodrug, copper-bis-belinostat (Cubisbel), in vitro and ex vivo, designed to overcome the pharmacokinetic challenges of belinostat. METHODS: The in vitro metabolism of each HDACi was evaluated in human liver microsomes (HLMs) using mass spectrometry. Next, the effect of belinostat and Cubisbel on cell growth, HDAC activity, apoptosis and cell cycle was assessed in three colon cancer cell lines. Gene expression alterations induced by both HDACis were determined using RNA-Seq, followed by in silico analysis to identify master regulators (MRs) of differentially expressed genes (DEGs). The effect of both HDACis on the viability of colon cancer patient-derived tumor organoids (PDTOs) was also examined. RESULTS: Belinostat and Cubisbel significantly reduced colon cancer cell growth mediated through HDAC inhibition and apoptosis induction. Interestingly, the in vitro half-life of Cubisbel was significantly longer than belinostat. Belinostat and its Cu derivative commonly dysregulated numerous signalling and metabolic pathways while genes downregulated by Cubisbel were potentially controlled by VEGFA, ERBB2 and DUSP2 MRs. Treatment of colon cancer PDTOs with the HDACis resulted in a significant reduction in cell viability and downregulation of stem cell and proliferation markers. CONCLUSIONS: Complexation of belinostat to Cu(II) does not alter the HDAC activity of belinostat, but instead significantly enhances its metabolic stability in vitro and targets anti-cancer pathways by perturbing key MRs in colon cancer. Complexation of HDACis to a metal ion might improve the efficacy of clinically used HDACis in patients with colon cancer.

Predictive modelling of response to neoadjuvant therapy in HER2+ breast cancer.

HER2-positive (HER2+) breast cancer accounts for 20-25% of all breast cancers. Predictive biomarkers of neoadjuvant therapy response are needed to better identify patients with early stage disease who may benefit from tailored treatments in the adjuvant setting. As part of the TCHL phase-II clinical trial (ICORG10-05/NCT01485926) whole exome DNA sequencing was carried out on normal-tumour pairs collected from 22 patients. Here we report predictive modelling of neoadjuvant therapy response using clinicopathological and genomic features of pre-treatment tumour biopsies identified age, estrogen receptor (ER) status and level of immune cell infiltration may together be important for predicting response. Clonal evolution analysis of longitudinally collected tumour samples show subclonal diversity and dynamics are evident with potential therapy resistant subclones detected. The sources of greater pre-treatment immunogenicity associated with a pathological complete response is largely unexplored in HER2+ tumours. However, here we point to the possibility of APOBEC associated mutagenesis, specifically in the ER-neg/HER2+ subtype as a potential mediator of this immunogenic phenotype.

Master Transcription Regulators and Transcription Factors Regulate Immune-Associated Differences Between Patients of African and European Ancestry With Colorectal Cancer.

Background and Aims: Individuals of African (AFR) ancestry have a higher incidence of colorectal cancer (CRC) than those of European (EUR) ancestry and exhibit significant health disparities. Previous studies have noted differences in the tumor microenvironment between AFR and EUR patients with CRC. However, the molecular regulatory processes that underpin these immune differences remain largely unknown. Methods: Multiomics analysis was carried out for 55 AFR and 456 EUR patients with microsatellite-stable CRC using The Cancer Genome Atlas. We evaluated the tumor microenvironment by using gene expression and methylation data, transcription factor, and master transcriptional regulator analysis to identify the cell signaling pathways mediating the observed phenotypic differences. Results: We demonstrate that downregulated genes in AFR patients with CRC showed enrichment for canonical pathways, including chemokine signaling. Moreover, evaluation of the tumor microenvironment showed that cytotoxic lymphocytes and neutrophil cell populations are significantly decreased in AFR compared with EUR patients, suggesting AFR patients have an attenuated immune response. We further demonstrate that molecules called "master transcriptional regulators" (MTRs) play a critical role in regulating the expression of genes impacting key immune processes through an intricate signal transduction network mediated by disease-associated transcription factors (TFs). Furthermore, a core set of these MTRs and TFs showed a positive correlation with levels of cytotoxic lymphocytes and neutrophils across both AFR and EUR patients with CRC, thus suggesting their role in driving the immune infiltrate differences between the two ancestral groups. Conclusion: Our study provides an insight into the intricate regulatory landscape of MTRs and TFs that orchestrate the differences in the tumor microenvironment between patients with CRC of AFR and EUR ancestry.

An Insight Into the Driver Mutations and Molecular Mechanisms Underlying Mucinous Adenocarcinoma of the Rectum.

BACKGROUND: Mucinous adenocarcinoma of the rectum accounts for 10% of all rectal cancers and has an impaired response to neoadjuvant chemoradiotherapy and worse overall survival. To date, insufficient genomic research has been performed on this histological subtype. OBJECTIVE: This study aims to define the mismatch repair deficiency rate and the driver mutations underpinning mucinous adenocarcinoma of the rectum and to compare it with rectal adenocarcinoma not otherwise specified. DESIGN: Immunohistochemistry and sequencing were performed on tumor samples from our tumor biobank. SETTINGS: This study was conducted across 2 tertiary referral centers. PATIENTS: Patients with mucinous adenocarcinoma and rectal adenocarcinoma not otherwise specified who underwent rectal resection between 2008 and 2018 were included. MAIN OUTCOME MEASURES: Mismatch repair status was performed by immunohistochemical staining. Mutations in the panel of oncogenes and tumor suppressor genes were determined by sequencing on the MiSeq V3 platform. RESULTS: The study included 33 patients with mucinous adenocarcinoma of the rectum and 100 patients with rectal adenocarcinoma not otherwise specified. Those with mucinous adenocarcinoma had a mismatch repair deficiency rate of 12.1% compared to 2.0% in the adenocarcinoma not otherwise specified cohort (p = 0.04). Mucinous adenocarcinoma and adenocarcinoma not otherwise specified rectal tumors had similar mutation frequencies in most oncogenes and tumor suppressor genes. No difference was found in the KRAS mutation rate (50.0% vs 37.1%, p = 0.29) or BRAF mutation rate (6.7% vs 3.1%, p = 0.34) between the cohorts. No difference was found between the cohorts regarding recurrence-free (p = 0.29) or overall survival (p = 0.14). LIMITATIONS: The major limitations of this study were the use of formalin-fixed, paraffin-embedded tissue over fresh-frozen tissue and the small number of patients included, in particular, in the mucinous rectal cohort. CONCLUSIONS: Most mucinous rectal tumors develop and progress along the chromosomal instability pathway. Further research in the form of transcriptomics, proteomics, and analysis of the effects of the mucin barrier may yield valuable insights into the mechanisms of resistance to chemoradiotherapy in this cohort. See Video Abstract at http://links.lww.com/DCR/B464. UNA PERCEPCIN SOBRE MUTACIONES IMPULSORAS Y MECANISMOS MOLECULARES SUBYACENTES AL ADENOCARCINOMA MUCINOSO DEL RECTO: ANTECEDENTES:El adenocarcinoma mucinoso del recto, representa el 10% de todos los cánceres rectales y tiene una respuesta deficiente a la quimioradioterapia neoadyuvante y una peor supervivencia en general. A la fecha, se han realizado muy pocas investigaciones genómicas sobre este subtipo histológico.OBJETIVO:Definir la tasa de deficiencia en la reparación de desajustes y mutaciones impulsoras, que sustentan el adenocarcinoma mucinoso del recto y compararlo con el adenocarcinoma rectal no especificado de otra manera.DISEÑO:Se realizaron inmunohistoquímica y secuenciación en muestras tumorales de nuestro biobanco de tumores.AJUSTE:El estudio se realizó en dos centros de referencia terciarios.PACIENTES:Se incluyeron pacientes con adenocarcinoma mucinoso y adenocarcinoma no especificado de otra manera, sometidos a resección rectal entre 2008 y 2018.PRINCIPALES MEDIDAS DE RESULTADO:El estado de reparación de desajustes se realizó mediante tinción inmunohistoquímica. Las mutaciones en el panel de oncogenes y genes supresores de tumores, se determinaron mediante secuenciación en la plataforma MiSeq V3.RESULTADOS:El estudio incluyó a 33 pacientes con adenocarcinoma mucinoso del recto y 100 pacientes con adenocarcinoma del recto no especificado de otra manera. Aquellos con adenocarcinoma mucinoso, tenían una tasa de deficiencia de reparación de desajustes del 12,1% en comparación con el 2,0% en la cohorte de adenocarcinoma no especificado de otra manera (p = 0,04). El adenocarcinoma mucinoso y el adenocarcinoma no especificado de otra manera, tuvieron frecuencias de mutación similares en la mayoría de los oncogenes y genes supresores de tumores. No se encontraron diferencias en la tasa de mutación de KRAS (50,0% frente a 37,1%, p = 0,29) o la tasa de mutación de BRAF (6,7% frente a 3,1%, p = 0,34) entre las cohortes. No se encontraron diferencias entre las cohortes con respecto a la supervivencia libre de recurrencia (p = 0,29) o la supervivencia global (p = 0,14).LIMITACIONES:Las mayores limitaciones de este estudio, fueron el uso de tejido embebido en parafina y fijado con formalina, sobre el tejido fresco congelado y el pequeño número de pacientes incluidos, particularmente en la cohorte mucinoso rectal.CONCLUSIONES:La mayoría de los tumores rectales mucinosos se desarrollan y progresan a lo largo de la vía de inestabilidad cromosómica. La investigación adicional en forma transcriptómica, proteómica y análisis de los efectos de la barrera de la mucina, puede proporcionar información valiosa sobre los mecanismos de resistencia a la quimioradioterapia, en esta cohorte. Consulte Video Resumen en http://links.lww.com/DCR/B464.

A Functional Genomic Screen Identifies the Deubiquitinase USP11 as a Novel Transcriptional Regulator of ERα in Breast Cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle-associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. SIGNIFICANCE: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.