Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma.

Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs. SIGNIFICANCE: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors.

Functional analysis of recurrent CDC20 promoter variants in human melanoma.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

Characterization of cell-type specific circular RNAs associated with colorectal cancer metastasis.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy and a leading cause of cancer deaths in the United States. More than half of CRC patients develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs) have recently emerged as important tumorigenesis regulators; however, their role in mCRC progression remains poorly characterized. Further, little is known about their cell-type specificity to elucidate their functions in the tumor microenvironment (TME). To address this, we performed total RNA sequencing (RNA-seq) on 30 matched normal, primary and metastatic samples from 14 mCRC patients. Additionally, five CRC cell lines were sequenced to construct a circRNA catalog in CRC. We detected 47 869 circRNAs, with 51% previously unannotated in CRC and 14% novel candidates when compared to existing circRNA databases. We identified 362 circRNAs differentially expressed in primary and/or metastatic tissues, termed circular RNAs associated with metastasis (CRAMS). We performed cell-type deconvolution using published single-cell RNA-seq datasets and applied a non-negative least squares statistical model to estimate cell-type specific circRNA expression. This predicted 667 circRNAs as exclusively expressed in a single cell type. Collectively, this serves as a valuable resource, TMECircDB (accessible at https://www.maherlab.com/tmecircdb-overview), for functional characterization of circRNAs in mCRC, specifically in the TME.

Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin.

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.

Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Neoadjuvant FOLFIRINOX Therapy Is Associated with Increased Effector T Cells and Reduced Suppressor Cells in Patients with Pancreatic Cancer.

PURPOSE: FOLFIRINOX has demonstrated promising results for patients with pancreatic ductal adenocarcinoma (PDAC). Chemotherapy-induced immunogenic cell death can prime antitumor immune responses. We therefore performed high-dimensional profiling of immune cell subsets in peripheral blood to evaluate the impact of FOLFIRINOX on the immune system. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells (PBMC) were obtained from treatment-naïve (n = 20) and FOLFIRINOX-treated patients (n = 19) with primary PDAC tumors at the time of resection. PBMCs were characterized by 36 markers using mass cytometry by time of flight (CyTOF). RESULTS: Compared with treatment-naïve patients, FOLFIRINOX-treated patients showed distinct immune profiles, including significantly decreased inflammatory monocytes and regulatory T cells (Treg), increased Th1 cells, and decreased Th2 cells. Notably, both monocytes and Treg expressed high levels of immune suppression-associated CD39, and the total CD39+ cell population was significantly lower in FOLFIRINOX-treated patients compared with untreated patients. Cellular alterations observed in responders to FOLFIRINOX included a significantly decreased frequency of Treg, an increased frequency of total CD8 T cells, and an increased frequency of CD27-Tbet+ effector/effector memory subsets of CD4 and CD8 T cells. CONCLUSIONS: Our study reveals that neoadjuvant chemotherapy with FOLFIRINOX enhances effector T cells and downregulates suppressor cells. These data indicate that FOLFIRINOX neoadjuvant therapy may improve immune therapy and clinical outcome in patients with PDAC.

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.

Genetic heterogeneity of induced pluripotent stem cells: results from 24 clones derived from a single C57BL/6 mouse.

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs "captures" the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically "fit" for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0-45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40-60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.