Highlights d CRISPR/Cas9 domain screen reveals RBP dependencies in AML d RBM39 is required for AML maintenance through missplicing of HOXA9 target genes d Proteomic studies identify an essential RBP splicing network in AML d Pharmacologic RBM39 degradation leads to broad antileukemic effects

Cellular transformation is accompanied by extensive re-wiring 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.

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-lm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the "single file" pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.

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.

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.

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tumor trafficking, and the formation of a tumor cell/T-cell immune response. This type of immunogenicity is comprised of the release of immune modulating signals, which include the release of high mobility group box 1 (HMGB1), ATP, and the translocation of calreticulin to the cell surface. Previous studies have demonstrated a positive correlation between markers of immunogenic cell death (ICD) and radiation dose with X-rays. Less is known about the effect of higher energies using charged particle irradiation and ICD. In this study, we explore whether varying linear energy transfer (LET) greater than 60 keV/mm with helium ion irradiation would impact immunogenicity in tumor cells as measured by levels of calreticulin translocation, ATP release, and HMGB1 release. Materials/Methods: Reporter cell lines designed to detect ATP luminescence (RLUs), red fluorescence protein tagged HMGB1 (relative fluorescent units [RFUs]), and cell surface calreticulin (percentage of calreticulin positive cells [%CRT+]) were derived from TSA (murine mammary carcinoma; syngeneic to BALB/c mice) cells. Prior to irradiation, the cells were seeded on six micron thick Mylar attached to 1.5 cm metal rings. The cells were then irradiated at the Radiological Research Accelerator Facility (RARAF) through a Track Segment Charged-Particle Irradiator. ATP and HMGB1 levels were measured 72 hours after RT and calreticulin was measured 24 hours after RT. Various doses of 250kV X-rays and LETs of helium ions were delivered and levels of these tumor ICD markers were determined. Results: For ATP measurements, when compared to non-irradiated controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in RLU fold-changes of 1.00 AE 0.05 (non-irradiated), 2.33 AE 0.08, 3.27 AE 0.19, 3.24 AE 0.10, 3.99 AE 0.33, 4.12 AE 0.32, and 2.91 AE 0.16 respectively. For HMGB1 measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in RFU fold-changes of 1.00 AE 0.02 (non-irradiated), 1.43 AE 0.03, 1.72 AE 0.03, 1.97 AE 0.06, 1.64 AE 0.03, 1.61 AE 0.04, and 1.51 AE 0.03, respectively. For calreticulin measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in %CRT+ fold-changes of 1.00 AE 0.12 (non-irradiated), 2.32 AE 0.15, 3.07 AE 0.32, 3.16 AE 0.17, 3.01 AE 0.12, 2.74 AE 0.39, and 1.61 AE 0.15, respectively. Conclusion: Immunogenicity of tumor cells as measured by immune cell death signals increases with high LET ion irradiation when compared to low LET X-ray irradiation at the same dose. These marker levels appear to decrease at LETs greater than 110 keV/mm. Further investigation is warranted on the effects of high LET charged particle irradiation on the tumor immune response.

T cell activation and exhaustion are associated with highly coordinated changes in gene expression and chromatin accessibility. While the contribution of key, state-specific transcription factors to these processes has been extensively explored, the role for chromatin regulatory complexes remains less clear. The mammalian SWI/SNF (mSWI/SNF) complexes are a diverse family of ATP-dependent remodeling complexes that regulate DNA accessibility and hence facilitate timely and appropriate gene expression. Recent genome-scale CRISPR screens have highlighted the involvement of mSWI/SNF complexes in the acquisition of divergent T cell states; however, the mechanisms by which these complexes regulate T cell activation and exhaustion and how such activities may be leveraged for therapeutic benefit remain poorly understood. Here, we developed an in vitro system to map chromatin-level changes occurring during acute and chronic TCR stimulation in human T cells and to investigate the contribution of the mSWI/SNF complexes throughout these processes. By gene expression and chromatin profiling, we show that our in vitro model closely recapitulates the T cell activation and exhaustion phenotypes observed in human tumors. We demonstrate that the mSWI/SNF complexes contribute to shaping the epigenome of T cells at different activation and exhaustion stages, directing subsequent changes in gene expression. Specifically, deletion of components demarcating the cBAF subcomplex within the mSWI/SNF family improves T cell survival and proliferation and leads to the down-regulation of exhaustion hallmarks. Notably, by using four different compounds that either degrade or inhibit the catalytic activity of SMARCA2 and SMARCA4, we demonstrate that mSWI/SNF disruption significantly increases human T cell fitness, decreases exhaustion and is associated to memory-like hallmarks in chronically stimulated human T cells. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T fitness and expansion and results in improved anti-tumor control in vivo. These findings reveal a central role for mSWI/SNF complexes in the processes of T cell activation and exhaustion, and suggest SMARCA4/2 ATPase inhibitors and degraders as potential strategies to enhance current T-cell based cancer immunotherapies. Citation Format: Elena Battistello, Kimberlee Hixon, Dawn E. Comstock, Clayton K. Collings, Xufeng Chen, Javier Rodriguez Hernaez, Soobeom Lee, Konstantinos Ntatsoulis, Annamaria Cesarano, Kathryn Hockemeyer, W. Nicholas Haining, Matthew T. Witkowski, Jun Qi, Aristotelis Tsirigos, Fabiana Perna, Ioannis Aifantis, Cigall Kadoch. Targeting mammalian SWI/SNF chromatin remodeling complexes to improve T cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1794.