Integrative CRISPR Activation and Small Molecule Inhibitor Screening for lncRNA Mediating BRAF Inhibitor Resistance in Melanoma.

The incidence of melanoma, being one of the most commonly occurring cancers, has been rising since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. The conventional targeted therapy is well defined and effective for advanced-stage melanomas for patients not responding to the standard-of-care immunotherapy. However, targeted therapies do not prove to be as effective as patients inevitably develop V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-inhibitor resistance to the respective drugs. Factors which are driving melanoma drug resistance mainly involve mutations in the mitogen-activated protein kinase (MAPK) pathway, e.g., BRAF splice variants, neuroblastoma RAS viral oncogene homolog (NRAS) amplification or parallel survival pathways. However, those mechanisms do not explain all cases of occurring resistances. Therefore, other factors accounting for BRAFi resistance must be better understood. Among them there are long non-coding RNAs (lncRNAs), but these remain functionally poorly understood. Here, we conduct a comprehensive, unbiased, and integrative study of lncRNA expression, coupled with a Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-mediated activation (CRISPRa) and small molecule inhibitor screening for BRAF inhibitor resistance to expand the knowledge of potentially druggable lncRNAs, their function, and pave the way for eventual combinatorial treatment approaches targeting diverse pathways in melanoma.

Stepwise activities of mSWI/SNF family chromatin remodeling complexes direct T cell activation and exhaustion.

Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.

Stereotactic body radiation therapy for an unresectable FGF23-secreting tumor of the cervical spine: A case report and literature review.

We present the case of a 65-year-old male with tumor-induced osteomalacia (TIO) caused by an FGF23-secreting phosphaturic tumor of C2 treated definitively with stereotactic body radiation therapy (SBRT) and kyphoplasty. The patient exhibited notable reduction in FGF23 6 weeks following radiotherapy. He also received a dose of the FGF23 monoclonal antibody, burosumab. We discuss the case with emphasis on radiation in the management of TIO. This case demonstrates SBRT as a well-tolerated local treatment option for the management of unresectable FGF23-producing tumors.

Siah2 control of T-regulatory cells limits anti-tumor immunity.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

Targeting an RNA-Binding Protein Network in Acute Myeloid Leukemia.

RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.

Oncogenic hijacking of the stress response machinery in T cell acute lymphoblastic leukemia.

Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

Study of Chemotaxis and Cell-Cell Interactions in Cancer with Microfluidic Devices.

Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video microscopy we can examine the chemotactic responses of neutrophil-like cells under conditions of varying gradient steepness or flow rate and then utilize software programs to calculate the speed and angles of cell migration as gradient steepness and flow are varied. Considering the shearing force generated on the cells by the constant flow that is required to produce and maintain a stable gradient, the trajectories of the cell migration will reflect the net result of both shear force generated by flow and the chemotactic force resulting from the chemokine gradient. Moreover, the effects of mutations in chemokine receptors or the presence of inhibitors of intracellular signals required for gradient sensing can be evaluated in real time. We also describe a method to monitor intracellular signals required for cells to alter cell polarity in response to an abrupt switch in gradient direction. Lastly, we demonstrate an in vitro method for studying the interactions of human cancer cells with human endothelial cells, fibroblasts, and leukocytes, as well as environmental chemokines and cytokines, using 3D microbioreactors that mimic the in vivo microenvironment.