SMARCA4 mutation induces tumor cell-intrinsic defects in enhancer landscape and resistance to immunotherapy.

Cancer genomic studies have identified frequent alterations in components of the SWI/SNF (SWItch/Sucrose Non- Fermenting) chromatin remodeling complex including SMARCA4 and ARID1A . Importantly, clinical reports indicate that SMARCA4 -mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. However, the mechanistic basis of immunotherapy resistance is unknown. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring SMARCA4 deficiency. Specifically, we show that SMARCA4 loss caused decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells (DCs) and CD4+ T cells into the tumor microenvironment (TME). Mechanistically, we show that SMARCA4 loss in tumor cells led to profound downregulation of STING, IL1ß and other components of the innate immune system as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. We establish that this deregulation of gene expression is caused by cancer cell-intrinsic reprogramming of the enhancer landscape with marked loss of chromatin accessibility at enhancers of genes involved in innate immune response such as STING, IL1ß, type I IFN and inflammatory cytokines. Interestingly, we observed that transcription factor NF-κB binding motif was highly enriched in enhancers that lose accessibility upon SMARCA4 deficiency. Finally, we confirmed that SMARCA4 and NF-κB co-occupy the same genomic loci on enhancers associated with STING and IL1ß, indicating a functional interplay between SMARCA4 and NF-κB. Taken together, our findings provide the mechanistic basis for the poor response of SMARCA4 -mutant tumors to anti-PD1 immunotherapy and establish a functional link between SMARCA4 and NF-κB on innate immune and inflammatory gene expression regulation.

Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze-Thaw Method.

Understanding the structure-function of inclusion bodies (IBs) in the last two decades has led to the development of several mild solubilization buffers for the improved recovery of bioactive proteins. The recently developed freeze-thaw-based inclusion body protein solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness. The present report investigates the reproducibility, efficiency, and plausible mechanism of the freeze-thaw-based IB solubilization. The percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in Escherichia coli and the quality attributes associated with the freeze-thaw-based solubilization method were analyzed in detail. The overall yield of the purified hGH and L-asparaginase protein was found to be around 14 and 25%, respectively. Both purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analyses revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides insight into the advantages, disadvantages, and molecular-structural information associated with the freeze-thaw-based solubilization method.

MN1 overexpression with varying tumor grade is a promising predictor of survival of glioma patients.

Gliomas have substantial mortality to incidence rate ratio and a dismal clinical course. Newer molecular insights, therefore, are imperative to refine glioma diagnosis, prognosis and therapy. Meningioma 1 (MN1) gene is a transcriptional co-regulator implicated in other malignancies, albeit its significance in glioma pathology remains to be explored. IGFBP5 is regulated transcriptionally by MN1 and IGF1 and is associated with higher glioma grade and shorter survival time, prompting us to ascertain their correlation in these tumors. We quantified the expression of MN1, IGFBP5 and IGF1 in 40 glioma samples and examined their interrelatedness. MN1 mRNA-protein inter-correlation and the gene's copy number were evaluated in these tumors. Publicly available TCGA datasets were used to examine the association of MN1 expression levels with patient survival and for validating our findings. We observed MN1 overexpression correlated with low-grade (LGGs) and not high-grade gliomas and is not determined by the copy number alteration of the gene. Notably, gliomas with upregulated MN1 have better overall survival (OS) and progression-free survival (PFS). IGFBP5 expression associated inversely with MN1 expression levels in gliomas but correlated positively with IGF1 expression in only LGGs. This suggests a potential grade-specific interplay between repressive and activating roles of MN1 and IGF1, respectively, in the regulation of IGFBP5. Thus, MN1 overexpression, a promising predictor of OS and PFS in gliomas, may serve as a prognostic biomarker in clinical practice to categorize patients with survival advantage.