Intermittent MEK inhibition with GITR co-stimulation rescues T-cell function for increased efficacy with CTLA-4 blockade in solid tumor models.

MEK inhibitors (MEKis) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression on tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T cell-receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and co-stimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor draining lymph nodes and tumors and led to intratumoral regulatory T cell (Treg) destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

APR-246 increases tumor antigenicity independent of p53.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

Evidence for a novel, effective approach to targeting carcinoma catabolism exploiting the first-in-class, anti-cancer mitochondrial drug, CPI-613.

Clinical targeting of the altered metabolism of tumor cells has long been considered an attractive hypothetical approach. However, this strategy has yet to perform well clinically. Metabolic redundancy is among the limitations on effectiveness of many approaches, engendering intrinsic single-agent resistance or efficient evolution of such resistance. We describe new studies of the multi-target, tumor-preferential inhibition of the mitochondrial tricarboxylic acid (TCA) cycle by the first-in-class drug CPI-613® (devimistat). By suppressing the TCA hub, indispensable to many metabolic pathways, CPI-613 substantially reduces the effective redundancy of tumor catabolism. This TCA cycle suppression also engenders an apparently homeostatic accelerated, inefficient consumption of nutrient stores in carcinoma cells, eroding some sources of drug resistance. Nonetheless, sufficiently abundant, cell line-specific lipid stores in carcinoma cells are among remaining sources of CPI-613 resistance in vitro and during the in vivo pharmacological drug pulse. Specifically, the fatty acid beta-oxidation step delivers electrons directly to the mitochondrial electron transport system (ETC), by-passing the TCA cycle CPI-613 target and producing drug resistance. Strikingly, tested carcinoma cell lines configure much of this fatty acid flow to initially traverse the peroxisome enroute to additional mitochondrial beta-oxidation. This feature facilitates targeting as clinically practical agents disrupting this flow are available. Two such agents significantly sensitize an otherwise fully CPI-613-resistant carcinoma xenograft in vivo. These and related results are strong empirical support for a potentially general class of strategies for enhanced clinical targeting of carcinoma catabolism.

Linear Desferrichrome-Linked Silicon-Rhodamine Antibody Conjugate Enables Targeted Multimodal Imaging of HER2 in Vitro and in Vivo.

We report the nuclear and optical in vitro and in vivo imaging of SKOV-3 cells by targeting HER2 with a bimodal trastuzumab conjugate. Previously, we have shown that desferrichrome derivatives provide a robust and versatile radiolabeling platform for the radioisotope zirconium-89. Here, we appended silicon-rhodamine functionalized linear desferrichrome to trastuzumab. This construct was radiolabeled and used to image cellular binding and antibody uptake in vitro and in vivo. The robust extinction coefficient of the SiR deep-red emissive fluorophore enables direct quantification of the number of appended chelators and fluorophore molecules per antibody. Subsequent radiolabeling of the multifunctional immunoconjugate with 89Zr was achieved with a 64 ± 9% radiochemical yield, while the reference immunoconjugate desferrioxamine (DFO)-trastuzumab exhibited a yield of 84 ± 9%. In vivo PET imaging (24, 48, 72, and 96 h post injection) and biodistribution experiments (96 h post injection) in HER2+ tumor bearing mice revealed no statistically significant difference of the two 89Zr-labeled conjugates at each time point evaluated. The bimodal conjugate permitted successful in vivo fluorescence imaging (96 h post injection) and subsequent fluorescence-guided, surgical resection of the tumor mass. This report details the first successful application of a fluorophore-functionalized desferrichrome derivative for targeted imaging, motivating further development and application of this scaffold as a multimodal imaging platform.