BET inhibitors rescue anti-PD1 resistance by enhancing TCF7 accessibility in leukemia-derived terminally exhausted CD8+ T cells.

Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.

HE4 Overexpression by Ovarian Cancer Promotes a Suppressive Tumor Immune Microenvironment and Enhanced Tumor and Macrophage PD-L1 Expression.

Ovarian cancer is a highly fatal malignancy characterized by early chemotherapy responsiveness but the eventual development of resistance. Immune targeting therapies are changing treatment paradigms for numerous cancer types but have had minimal success in ovarian cancer. Through retrospective patient sample analysis, we have determined that high human epididymis protein 4 (HE4) production correlates with multiple markers of immune suppression in ovarian cancer, including lower CD8+ T cell infiltration, higher PD-L1 expression, and an increase in the peripheral monocyte to lymphocyte ratio. To further understand the impact that HE4 has on the immune microenvironment in ovarian cancer, we injected rats with syngeneic HE4 high- and low-expressing cancer cells and analyzed the differences in their tumor and ascites immune milieu. We found that high tumoral HE4 expression promotes an ascites cytokine profile that is rich in myeloid-recruiting and differentiation factors, with an influx of M2 macrophages and increased arginase 1 production. Additionally, CTL activation is significantly reduced in the ascites fluid, and there is a trend toward lower CTL infiltration of the tumor, whereas NK cell recruitment to the ascites and tumor is also reduced. PD-L1 expression by tumor cells and macrophages is increased by HE4 through a novel posttranscriptional mechanism. Our data have identified HE4 as a mediator of tumor-immune suppression in ovarian cancer, highlighting this molecule as a potential therapeutic target for the treatment of this devastating disease.

RhoA/ROCK pathway inhibitor ameliorates erectile dysfunction induced by radiation therapy in rats.

OBJECTIVES: Prostate cancer (PCa) treatment with radiation therapy (RT) has an excellent cure rate. However, Radiation-induced Erectile Dysfunction (RiED) is a common and irreversible toxicity impacting quality of life, and there is no FDA approved specific drug for RiED. We previously showed that prostate RT increased RhoA/ROCK signaling in the cavernous nerve (CN) and penile tissues, which may lead to RiED in rats. In this study, we investigated whether RhoA/ROCK pathway inhibition by a specific inhibitor called Hydroxyfasudil (HF) can improve RiED in our well-established rat model. MATERIALS/METHODS: Male Sprague-Dawley rats were randomized to the following groups: sham-RT, HF-only, RT-only, and RT + HF. Rats were either exposed to a single dose of 25 Gy prostate-confined RT or a sham procedure. 10 mg/kg HF or normal saline was injected intraperitoneally. Erectile function was evaluated by intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements at week 14 post-RT. Cavernous nerve (CN) injury was evaluated by transmission electron microscopy (TEM), and penile tissue fibrosis by Masson trichrome staining (MT). RESULTS: We have found that the HF treatment prior to RT showed significant (p < 0.001) improvement in ICP/MAP ratio, area under the curve, and maximum ICP value, compared to RT-alone rats. Furthermore, RT + HF treated rats exhibited increased CN myelination and decreased axonal atrophy, comparted to RT-only. HF treatment showed significantly decreased penile tissue fibrosis (p < 0.05) compared to RT-alone treated rats. CONCLUSION: Our results provide the first preclinical evidence that targeting RhoA/ROCK pathway by HF may provide a novel therapeutic option for the treatment of RiED.

Online dose delivery verification in small animal image-guided radiotherapy.

PURPOSE: To accomplish novel radiation treatment techniques in preclinical radiation research, small animal image-guided radiotherapy systems have been increasingly integrated into preclinical radiation research over the last decade. Although such systems have sophisticated tools (such as cone-beam computed tomography-based image guidance, robotic couch, treatment planning system (TPS), and electronic portal imaging devices [EPIDs]). To our knowledge, no established technique can perform independent and online verification of the delivered dose during radiotherapy. In this study, we implement an online EPID dosimetry for each administered SA-IGRT fraction in a rat orthotopic model of prostate cancer. METHODS: To verify the accuracy of delivered dose to rat, we compared the two-dimensional (2D) calculated dose distribution by TPS as the planned dose, with online dose distribution estimated using an EPID as the delivered dose. Since image acquisition software was not capable of acquiring integrated images over a long period of time, we used the EPID to estimate dose rate rather than dose. The central axis (CAX) dose rate values at the beam's exit surface were compared. In addition, 2D dose distributions were also compared under different gamma criteria. To verify the accuracy of our EPID dosimetry technique, we measured transit and exit doses with film during animal treatment. In this study, 20-mm cone was used to collimate beam. We previously observed that the EPID response was independent of collimator size for collimator size ≥15-mm, we did not apply for additional correction factor. RESULTS: Comparison of exit CAX dose rate values of TPS-calculated and EPID-estimated showed that the average difference was 3.1%, with a maximum of 9.3%. Results of gamma analysis for 2D comparison indicated an average of 90% passing rate with global gamma criterion of 2 mm/5%. We observed that TPS could not calculate dose accurately in peripheral regions in which the penumbra effect was dominant. Dose rate values estimated by EPID were within 2.1% agreement with film at both the imager plane and the beam's exit surface for 4 randomly selected animals for which film measurement verification was performed. CONCLUSIONS: The small animal radiation research platform (SARRP) system's built-in EPID was utilized to estimate dose delivered to rats at kilovoltage energy x-rays. The results of this study suggest that the EPID is an invaluable tool for verifying delivered dose to small animal to help validate conclusions made from preclinical radiation research.