Ex vivo assessment of targeted therapies in a rare metastatic epithelial–myoepithelial carcinoma

Epithelial–myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial–myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial–myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia

CDK‐mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC‐driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer

Effects of interferon on cellular proliferation and apoptosis

Interferon (IFN) therapy is today a well established treatment in many diseases, including various malignancies. How IFN exerts antitumor activity is not known, but several mechanisms have been suggested. Previous studies have shown a correlation between the in vitro susceptibility of primary malignant cells to IFN and the clinical response of the patient to IFN therapy, supporting the idea that the antitumor activity of IFN results from direct effects of IFN on the tumor cells. The studies in the present thesis have dealt with the cellular effects of IFN on both malignant and normal cells, with the focus on how IFN modulates proliferation and apoptosis. In addition to their well known antiviral effects, IFNs can exert pleiotropic effects on cells, including potent cell growth inhibition of many cell types. Studies on various tumor cell lines and normal cells, as well as primary tumor cells, have established the antiproliferative effect as an important contributor to the decreased number of tumor cells commonly observed following IFN treatment. The role of apoptosis with regard to IFN's anticellular effects has so far been poorly defined. Using as a model system a number of hematopoietic cell lines, we showed that IFN-[alpha] in vitro is a potent inducer of apoptotic cell death, and that IFN-[alpha] -mediated growth arrest and apoptosis are independent responses to IFN-[alpha]. IFN-[alpha] induces remissions in approximately 15% of patients with multiple myeloma. We have previously shown that IFN-[alpha] exerts a direct cytotoxic effect on myeloma cells from some patients. Analysis of expression of the apoptosis-inhibitory protein, Bcl-2, in pre-treatment bone-marrow samples from patients with myeloma revealed a significant association (p=0.012) between high levels of Bcl-2 and resistance to IFN-[alpha] therapy. These data indicate that over-expression of Bcl-2 may be a cause for resistance to IFN-[alpha] therapy in myeloma, and that one possible mechanism for IFN's antitumor effect in this disease may be induction of apoptosis. In addition to these cytoreductive effects, IFNs have also in some systems been shown to protect malignant cells from apoptosis induced by different stimuli. In a study of p53-induced apoptosis, IFN-[gamma], but not IFN-[alpha], was found to protect cells from apoptosis. In other studies, the molecular mechanism behind IFN-[alpha] induced cell growth arrest was examined. The effect of IFN-[alpha] on expression of members of the cyclin-dependent kinase inhibitor (CKI) families was investigated in sensitive and resistant tumor cell lines, as well as in normal IL-2 - stimulated T-cells. The results demonstrated that IFN-[alpha] is a potent regulator of several CK1s, both from the INK4 family (p15) and the CIP/KIP family (p21 and p27). In sensitive tumor cell lines, a primary response to IFN-[alpha] is induction and binding of p21 to the Gl cyclin dependent kinases (CDKS) CDK4 and CDK2, causing inhibition of these kinases. Secondary events include the increased expression and accumulation of p27 in these G1 CDK complexes, rather than loss of the Gl kinase cyclin components, as well as dephosphorylation of the different pocket proteins. Importantly, in a resistant cell line, p21 protein was not expressed, despite high levels of p21 mRNA following IFN-[alpha] treatment. In normal IL-2 -stimulated T-cells, IFN-[alpha] was found to prevent entry into S-phase, correlating with profound inhibition of IL-2 -induced changes in G 1 regulatory proteins, including the prevention of n-litogen-induced reduction of p27 levels and upregulation of G l cyclins and CDKS

Targeting SOX9 for degradation to inhibit chemoresistance, metastatic spread, and recurrence

Cancer cells with stem-like properties are believed to contribute to treatment resistance, dissemination, and recurrence. SOX9 controls stem cell plasticity and its deregulation may provide a basis for tumor progression. Here, we summarize our findings of targeted SOX9 destruction by SCFFBW7 (Skp1/Cul1/F-box) in medulloblastoma and its potential for therapeutic intervention

Disruption of a novel Ectodermal Neural Cortex 1 antisense gene, ENC- 1AS and identification of ENC-1 overexpression in hairy cell leukemia

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