Mutated forms of the RAS oncogene drive 30% of all cancers, but they cannot be targeted therapeutically using currently available drugs. The molecular and cellular mechanisms that create a heterogenous tumor environment harboring both mutant and wild-type RAS have not been elucidated. In this study, we examined horizontal transfer of mutant KRAS between colorectal cancer (CRC) cells via a direct form of cell-to-cell communication called tunneling nanotubes (TNTs). TNT formation was significantly higher in CRC cell lines expressing mutant KRAS than CRC cell lines expressing wild-type RAS; this effect was most pronounced in metastatic CRC cell lines with both mutant KRAS and deficiency in mismatch repair proteins. Using inverted and confocal fluorescence time-lapse and fluorescence recovery after photobleaching (FRAP)-based microscopy, we observed GFP-tagged mutant KRAS^G12D protein trafficking between CRC cells through TNTs within a span of seconds to several minutes. Notably, acquisition of mutant KRAS increased Extracellular Signal-regulated Kinase (ERK) phosphorylation and upregulated tunneling nanotube formation in recipient wildtype CRC cells. In conclusion, these findings suggest that intercellular horizontal transfer of RAS can occur by TNTs. We propose that intercellular transfer of mutant RAS can potentially induce intratumoral heterogeneity and result in a more invasive phenotype in recipient cells.

Nanocarriers have been extensively utilized for the systemic targeting of various solid tumors and their metastases. However, current drug delivery systems, in general, suffer from a lack of selectivity for tumor cells. Here, we develop a novel two-step targeting strategy that relies on the selective accumulation of targetable synthetic receptors (i.e., azide moieties) in tumor tissues, followed by delivery of drug-loaded nanoparticles having a high binding affinity for these receptors. Mesenchymal stem cells (MSCs) were used as vehicles for the tumor-specific accumulation of azide moieties, while dibenzyl cyclooctyne (DBCO) was used as the targeting ligand. Biodistribution and antitumor efficacy studies were performed in both orthotopic metastatic and patient-derived xenograft (PDX) tumor models of ovarian cancer. Our studies show that nanoparticles are retained in tumors at a significantly higher concentration in mice that received azide-labeled MSCs (MSC-Az). Furthermore, we observed significantly reduced tumor growth (p < 0.05) and improved survival in mice receiving MSC-Az along with paclitaxel-loaded DBCO-functionalized nanoparticles compared to controls. These studies demonstrate the feasibility of a two-step targeting strategy for efficient delivery of concentrated chemotherapy for treating solid tumors.

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.

The majority of patients with high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy; however, most will develop chemotherapy resistance. Gene signatures may change with the development of chemotherapy resistance in this population, which is important as it may lead to tailored therapies. The objective of this study was to compare tumor gene expression profiles in patients before and after treatment with neoadjuvant chemotherapy (NACT). Tumor samples were collected from six patients diagnosed with HGSOC before and after administration of NACT. RNA extraction and whole transcriptome sequencing was performed. Differential gene expression, hierarchical clustering, gene set enrichment analysis, and pathway analysis were examined in all of the samples. Tumor samples clustered based on exposure to chemotherapy as opposed to patient source. Pre-NACT samples were enriched for multiple pathways involving cell cycle growth. Post-NACT samples were enriched for drug transport and peroxisome pathways. Molecular subtypes based on the pre-NACT sample (differentiated, mesenchymal, proliferative and immunoreactive) changed in four patients after administration of NACT. Multiple changes in tumor gene expression profiles after exposure to NACT were identified from this pilot study and warrant further attention as they may indicate early changes in the development of chemotherapy resistance.

Chronic inflammation of the colon (colitis) is a known risk factor for inflammatory-driven colorectal cancers (id-CRCs), and intestinal microbiota has been implicated in the etiology of id-CRCs. Manipulation of the microbiome is a clinically viable therapeutic approach to limiting id-CRCs. To understand the microbiome changes that occur over time in id-CRCs, we used a mouse model of id-CRCs with the treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS) and measured the microbiome over time. We included cohorts where the microbiome was restored using cage bedding swapping and where the microbiome was depleted using antibiotics to compare to untreated animals. We identified consistent increases in Akkermansia in mice receiving horizontal microbiome transfer (HMT) via cage bedding swapping, while the control cohort had consistent longitudinal increases in Anaeroplasma and Alistipes. Additionally, fecal lipocalin-2 (Lcn-2), a marker of intestinal inflammation, was elevated in unrestored animals compared to restored and antibiotic-treated counterparts following HMT. These observations suggest a potential role for Akkermansia, Anaeroplasma, and Alistipes in regulating colonic inflammation in id-CRCs.

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant patients with ovarian cancer is forthcoming. ARID1A-mutated ovarian clear cell carcinoma (OCCC) is a relatively indolent and chemoresistant ovarian cancer histotype. In OCCC-derived cells ARID1A simultaneously drives GLS1 expression and metabolism reprograming. In ARID1A-mutated OCCC-derived mouse models, loss of ARID1A corresponds to GLS1 upregulation and increases sensitivity to GLS1 inhibition. Thus, targeting of GLS1 with CB839 has been suggested as a targeted approach for patients with OCCC with tumors harboring ARID1A mutations. Here, we investigated whether GLS1 is differentially expressed between patients with OCCC whose tumors are ARID1A positive and patients whose tumors are ARID1A negative. In clinical specimens of OCCC, we found that GLS1 overexpression was not correlated with ARID1A loss. In addition, GLS1 overexpression was associated with better clinical outcomes. Our findings have implications for human trials using experimental therapeutics targeting GLS1.Significance: GLS1 differential expression in patients with OCCC with or without ARID1A mutations is significant because a clinical trial with a GLS1 inhibitor is forthcoming. Tumors without ARID1A have low levels of GLS1 and GLS1 expression is associated to better outcome. Thus, blockade of GLS1 could be counterproductive for patients with OCCC.