Suramin protects from cisplatin-induced acute kidney injury.

Cisplatin, a commonly used cancer chemotherapeutic, has a dose-limiting side effect of nephrotoxicity. Approximately 30% of patients administered cisplatin suffer from kidney injury, and there are limited treatment options for the treatment of cisplatin-induced kidney injury. Suramin, which is Federal Drug Administration-approved for the treatment of trypanosomiasis, improves kidney function after various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin-induced kidney injury. Suramin treatment before cisplatin administration reduced cisplatin-induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin-induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 h after cisplatin also improved kidney function, suggesting that the mechanism of protection is not by inhibition of tubular cisplatin uptake or its metabolism to nephrotoxic species. If suramin is to be used in the context of cancer, then it cannot prevent cisplatin-induced cytotoxicity of cancer cells. Suramin did not alter the dose-response curve of cisplatin in lung adenocarcinoma cells in vitro. In addition, suramin pretreatment of mice harboring lung adenocarcinomas did not alter the initial cytotoxic effects of cisplatin (DNA damage and apoptosis) on tumor cells. These results provide evidence that suramin has potential as a renoprotective agent for the treatment/prevention of cisplatin-induced acute kidney injury and justify future long-term preclinical studies using cotreatment of suramin and cisplatin in mouse models of cancer.

Inhibition of ULK1/2 and KRAS G12C controls tumor growth in preclinical models of lung cancer.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent FDA approval of covalent inhibitors of KRAS G12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRAS G12C -driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients that do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRAS G12C , efforts are underway to develop effective combination therapies. Here we report that inhibition of KRAS G12C signaling increases autophagy in KRAS G12C expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRAS G12C -driven lung cancer cell proliferation in vitro and superior tumor control in vivo . Additionally, in genetically engineered mouse models of KRAS G12C -driven NSCLC, inhibition of either KRAS G12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRAS G12C in lung cancer.

Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis.

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/ß-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression. SIGNIFICANCE: Loss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

Inhibition of MEK1/2 Forestalls the Onset of Acquired Resistance to Entrectinib in Multiple Models of NTRK1-Driven Cancer.

NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.

Chloroquine Sensitizes GNAQ/11-mutated Melanoma to MEK1/2 Inhibition.

PURPOSE: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma. EXPERIMENTAL DESIGN: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity. RESULTS: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death. CONCLUSIONS: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties.

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

The PIM family of oncoproteins: small kinases with huge implications in myeloid leukemogenesis and as therapeutic targets.

PIM kinases are a family of serine/threonine kinases involved in cell survival and proliferation. There is significant structural similarity between the three PIM kinases (PIM1, PIM2 and PIM3) and only few amino acid differences. Although, several studies have specifically monitored the role of PIM1 in tumorigenesis, much less is known about PIM2 and PIM3. Therefore, in this study we have used in vitro cell culture models and in vivo bone marrow infection/transplantation to assess the comparative signaling and oncogenic potential of each of the three PIM kinases. All three PIM kinases were able to protect FL5.12 cells from IL3 withdrawal induced death. Interestingly, the downstream signaling cascades were indistinguishable between the three kinases. Transplantation of murine bone marrow co-expressing MYC and PIM1, PIM2 or PIM3 caused rapid and uniformly lethal myeloid leukemia. De-induction of MYC 18 days following transplantation significantly increased the survival of mice, even with continual expression of PIM kinases. Alternatively, mice treated at the pre-leukemic stage with a PIM kinase inhibitor increased the lifespan of the mice, even with continual expression of the MYC transgene. These data demonstrate the role of PIM kinases in driving myeloid leukemia, and as candidate molecules for therapy against human malignancies.

Dissecting the in vivo leukemogenic potency of BCLxl.

Overexpression of anti-apoptotic members of the BCL2 family has been found in all types of cancer. A member of the family, BCLxl (B-cell lymphoma extra-large), is known to be associated with the progression of leukemogenesis. In the present study, we focused on understanding the domains of BCLxl responsible for in vivo oncogenic potency. To this end, we utilized engineered BCLxl proteins with alternative transmembrane domains (TM) or chimeric BCLxl proteins containing domains from a less potent BCL2-like protein, BCLb. As expected, mice receiving MYC-only expressing bone marrow develop leukemia by 100 days, whereas co-expression of MYC with wild-type BCLxl led to aggressive myeloid leukemia with an average latency of ~25 days. Interestingly, mice injected with bone marrow co-expressing MYC and BCLxl targeted specifically to either mitochondria or ER also succumbed to leukemia with an average latency of ~25 days. Further, our study was extended to examine the role of the BH4 domain in driving potent leukemogenesis. Mice injected with bone marrow co-expressing MYC and BCLb succumb to leukemia in an average of ~55 days, but interestingly a BCLxl protein containing only the loop region of BCLb drove MYC-induced leukemogenesis with the same latency as wild-type BCLxl. These data suggest that the localization of exogenous BCLxl to either mitochondria or ER is not a steadfast dictator of in vivo oncogenic potency. Further, our findings suggest that the loop domain of BCLb and BCLxl is not responsible for dictating the in vivo leukemogeneic potency. This study provides further mechanistic details into the biochemical functions of BCLxl.