Cancer treatments as paradoxical catalysts of tumor awakening in the lung.

Much of the fatality of tumors is linked to the growth of metastases, which can emerge months to years after apparently successful treatment of primary tumors. Metastases arise from disseminated tumor cells (DTCs), which disperse through the body in a dormant state to seed distant sites. While some DTCs lodge in pre-metastatic niches (PMNs) and rapidly develop into metastases, other DTCs settle in distinct microenvironments that maintain them in a dormant state. Subsequent awakening, induced by changes in the microenvironment of the DTC, causes outgrowth of metastases. Hence, there has been extensive investigation of the factors causing survival and subsequent awakening of DTCs, with the goal of disrupting these processes to decrease cancer lethality. We here provide a detailed overview of recent developments in understanding of the factors controlling dormancy and awakening in the lung, a common site of metastasis for many solid tumors. These factors include dynamic interactions between DTCs and diverse epithelial, mesenchymal, and immune cell populations resident in the lung. Paradoxically, among key triggers for metastatic outgrowth, lung tissue remodeling arising from damage induced by the treatment of primary tumors play a significant role. In addition, growing evidence emphasizes roles for inflammation and aging in opposing the factors that maintain dormancy. Finally, we discuss strategies being developed or employed to reduce the risk of metastatic recurrence.

Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors. SIGNIFICANCE: The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

Musashi-2 (MSI2) regulation of DNA damage response in lung cancer.

Lung cancer is one of the most common types of cancer worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras-activating mutation and Trp53 deletion, with and without Msi2 deletion (KPM2 versus KP mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice. In addition, KPM2 lung tumors showed evidence of decreased proliferation, but increased DNA damage, marked by increased levels of phH2AX (S139) and phCHK1 (S345), but decreased total and activated ATM. Using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo. Taken together, we conclude that MSI2 supports NSCLC tumorigenesis, in part, by supporting repair of DNA damage by controlling expression of DDR proteins. These results suggest that targeting MSI2 may be a promising strategy for lung cancers treated with DNA-damaging agents.

Source, co-occurrence, and prognostic value of PTEN mutations or loss in colorectal cancer.

Somatic PTEN mutations are common and have driver function in some cancer types. However, in colorectal cancers (CRCs), somatic PTEN-inactivating mutations occur at a low frequency (~8-9%), and whether these mutations are actively selected and promote tumor aggressiveness has been controversial. Analysis of genomic data from ~53,000 CRCs indicates that hotspot mutation patterns in PTEN partially reflect DNA-dependent selection pressures, but also suggests a strong selection pressure based on protein function. In microsatellite stable (MSS) tumors, PTEN alterations co-occur with mutations activating BRAF or PI3K, or with TP53 deletions, but not in CRC with microsatellite instability (MSI). Unexpectedly, PTEN deletions are associated with poor survival in MSS CRC, whereas PTEN mutations are associated with improved survival in MSI CRC. These and other data suggest use of PTEN as a prognostic marker is valid in CRC, but such use must consider driver mutation landscape, tumor subtype, and category of PTEN alteration.

Trogocytosis of cancer-associated fibroblasts promotes pancreatic cancer growth and immune suppression via phospholipid scramblase anoctamin 6 (ANO6).

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

Loss of Pkd1 limits susceptibility to colitis and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~50,000 deaths. Autosomal dominant polycystic kidney disease (ADPKD), associated with mutations disabling the PKD1 gene, affects as many as 1 in 1000. Intriguingly, some studies have suggested that individuals with germline mutations in PKD1 have reduced incidence of CRC, suggesting a genetic modifier function. Using mouse models, we here establish that loss of Pkd1 greatly reduces CRC incidence and tumor growth induced by loss of the tumor suppressor Apc. Growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/- organoids, indicating a cancer cell-intrinsic activity, even though Pkd1 loss enhanced activity of pro-oncogenic signaling pathways. Notably, Pkd1 loss increased colon barrier function, with Pkd1-deficient animals resistant to DSS-induced colitis, associated with upregulation of claudins that decrease permeability, and reduced T cell infiltration. Notably, Pkd1 loss caused greater sensitivity to activation of CFTR, a tumor suppressor in CRC, paralleling signaling relations in ADPKD. Overall, these data and other data suggest germline and somatic mutations in PKD1 may influence incidence, presentation, and treatment response in human CRC and other pathologies involving the colon.

Musashi-2 (MSI2) regulation of DNA damage response in lung cancer.

Lung cancer is one of the most common types of cancers worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras -activating mutation and Trp53 deletion, with and without Msi2 deletion (KP versus KPM2 mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice what supports published data. In addition, using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM/Atm mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo . Taken together, we conclude that MSI2 supports lung tumorigenesis, in part, by direct positive regulation of ATM protein expression and DDR. This adds the knowledge of MSI2 function in lung cancer development. Targeting MSI2 may be a promising strategy to treat lung cancer. Significance: This study shows the novel role of Musashi-2 as regulator of ATM expression and DDR in lung cancer.

Intrapancreatic fat, pancreatitis, and pancreatic cancer.

Pancreatic cancer is typically detected at an advanced stage, and is refractory to most forms of treatment, contributing to poor survival outcomes. The incidence of pancreatic cancer is gradually increasing, linked to an aging population and increasing rates of obesity and pancreatitis, which are risk factors for this cancer. Sources of risk include adipokine signaling from fat cells throughout the body, elevated levels of intrapancreatic intrapancreatic adipocytes (IPAs), inflammatory signals arising from pancreas-infiltrating immune cells and a fibrotic environment induced by recurring cycles of pancreatic obstruction and acinar cell lysis. Once cancers become established, reorganization of pancreatic tissue typically excludes IPAs from the tumor microenvironment, which instead consists of cancer cells embedded in a specialized microenvironment derived from cancer-associated fibroblasts (CAFs). While cancer cell interactions with CAFs and immune cells have been the topic of much investigation, mechanistic studies of the source and function of IPAs in the pre-cancerous niche are much less developed. Intriguingly, an extensive review of studies addressing the accumulation and activity of IPAs in the pancreas reveals that unexpectedly diverse group of factors cause replacement of acinar tissue with IPAs, particularly in the mouse models that are essential tools for research into pancreatic cancer. Genes implicated in regulation of IPA accumulation include KRAS, MYC, TGF-ß, periostin, HNF1, and regulators of ductal ciliation and ER stress, among others. These findings emphasize the importance of studying pancreas-damaging factors in the pre-cancerous environment, and have significant implications for the interpretation of data from mouse models for pancreatic cancer.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.

BACKGROUND: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. METHODS: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. RESULTS: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. CONCLUSIONS: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

Patterns of Ciliation and Ciliary Signaling in Cancer.

Among the factors that have been strongly implicated in regulating cancerous transformation, the primary monocilium (cilium) has gained increasing attention. The cilium is a small organelle extending from the plasma membrane, which provides a localized hub for concentration of transmembrane receptors. These receptors transmit signals from soluble factors (including Sonic hedgehog (SHH), platelet-derived growth factor (PDGF-AA), WNT, TGFß, NOTCH, and others) that regulate cell growth, as well as mechanosensory cues provided by flow or extracellular matrix. Ciliation is regulated by cell cycle, with most cells that are in G0 (quiescent) or early G1 ciliation and cilia typically absent in G2/M cells. Notably, while most cells organized in solid tissues are ciliated, cancerous transformation induces significant changes in ciliation. Most cancer cells lose cilia; medulloblastomas and basal cell carcinomas, dependent on an active SHH pathway, rely on ciliary maintenance. Changes in cancer cell ciliation are driven by core oncogenic pathways (EGFR, KRAS, AURKA, PI3K), and importantly ciliation status regulates functionality of those pathways. Ciliation is both influenced by targeted cancer therapies and linked to therapeutic resistance; recent studies suggest ciliation may also influence cancer cell metabolism and stem cell identity. We review recent studies defining the relationship between cilia and cancer.

Aurora Kinases as Therapeutic Targets in Head and Neck Cancer.

ABSTRACT: The Aurora kinases (AURKA and AURKB) have attracted attention as therapeutic targets in head and neck squamous cell carcinomas. Aurora kinases were first defined as regulators of mitosis that localization to the centrosome (AURKA) and centromere (AURKB), governing formation of the mitotic spindle, chromatin condensation, activation of the core mitotic kinase CDK1, alignment of chromosomes at metaphase, and other processes. Subsequently, additional roles for Aurora kinases have been defined in other phases of cell cycle, including regulation of ciliary disassembly and DNA replication. In cancer, elevated expression and activity of Aurora kinases result in enhanced or neomorphic locations and functions that promote aggressive disease, including promotion of MYC expression, oncogenic signaling, stem cell identity, epithelial-mesenchymal transition, and drug resistance. Numerous Aurora-targeted inhibitors have been developed and are being assessed in preclinical and clinical trials, with the goal of improving head and neck squamous cell carcinoma treatment.

Clinical Trial Development in TP53-Mutated Locally Advanced and Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma.

TP53 mutation is the most frequent genetic event in head and neck squamous cell carcinoma (HNSCC), found in more than 80% of patients with human papillomavirus-negative disease. As mutations in the TP53 gene are associated with worse outcomes in HNSCC, novel therapeutic approaches are needed for patients with TP53-mutated tumors. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issues of identifying and developing clinical trials for patients with TP53 mutations. Subcommittees, or breakout groups, were tasked with developing clinical studies in both the locally advanced and recurrent and/or metastatic (R/M) disease settings as well as considering signal-seeking trial designs. A fourth breakout group was focused on identifying and standardizing biomarker integration into trial design; this information was provided to the other breakout groups prior to the meeting to aid in study development. A total of 4 concepts were prioritized to move forward for further development and implementation. This article summarizes the proceedings of the Clinical Trials Planning Meeting with the goal of developing clinical trials for patients with TP53-mutant HNSCC that can be conducted within the National Clinical Trials Network.

Delineating the RAS Conformational Landscape.

Mutations in RAS isoforms (KRAS, NRAS, and HRAS) are among the most frequent oncogenic alterations in many cancers, making these proteins high priority therapeutic targets. Effectively targeting RAS isoforms requires an exact understanding of their active, inactive, and druggable conformations. However, there is no structural catalog of RAS conformations to guide therapeutic targeting or examining the structural impact of RAS mutations. Here we present an expanded classification of RAS conformations based on analyses of the catalytic switch 1 (SW1) and switch 2 (SW2) loops. From 721 human KRAS, NRAS, and HRAS structures available in the Protein Data Bank (206 RAS-protein cocomplexes, 190 inhibitor-bound, and 325 unbound, including 204 WT and 517 mutated structures), we created a broad conformational classification based on the spatial positions of Y32 in SW1 and Y71 in SW2. Clustering all well-modeled SW1 and SW2 loops using a density-based machine learning algorithm defined additional conformational subsets, some previously undescribed. Three SW1 conformations and nine SW2 conformations were identified, each associated with different nucleotide states (GTP-bound, nucleotide-free, and GDP-bound) and specific bound proteins or inhibitor sites. The GTP-bound SW1 conformation could be further subdivided on the basis of the hydrogen bond type made between Y32 and the GTP γ-phosphate. Further analysis clarified the catalytic impact of G12D and G12V mutations and the inhibitor chemistries that bind to each druggable RAS conformation. Overall, this study has expanded our understanding of RAS structural biology, which could facilitate future RAS drug discovery. SIGNIFICANCE: Analysis of >700 RAS structures helps define an expanded landscape of active, inactive, and druggable RAS conformations, the structural impact of common RAS mutations, and previously uncharacterized RAS inhibitor-binding modes.

The role of NSD1, NSD2, and NSD3 histone methyltransferases in solid tumors.

NSD1, NSD2, and NSD3 constitute the nuclear receptor-binding SET Domain (NSD) family of histone 3 lysine 36 (H3K36) methyltransferases. These structurally similar enzymes mono- and di-methylate H3K36, which contribute to the maintenance of chromatin integrity and regulate the expression of genes that control cell division, apoptosis, DNA repair, and epithelial-mesenchymal transition (EMT). Aberrant expression or mutation of members of the NSD family is associated with developmental defects and the occurrence of some types of cancer. In this review, we discuss the effect of alterations in NSDs on cancer patient's prognosis and response to treatment. We summarize the current understanding of the biological functions of NSD proteins, focusing on their activities and the role in the formation and progression in solid tumors biology, as well as how it depends on tumor etiologies. This review also discusses ongoing efforts to develop NSD inhibitors as a promising new class of cancer therapeutic agents.

Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC. EXPERIMENTAL DESIGN: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations. RESULTS: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation. CONCLUSIONS: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.