Comment in Cancer Res. 2022 Sep 2;82(17):2977-2979.

Accumulating evidence has demonstrated that drug resistance can be acquired in cancer through the repopulation of tumors by cancer stem cell (CSC) expansion. Here, we investigated mechanisms driving resistance and CSC repopulation in hepatocellular carcinoma (HCC) as a cancer model using two drug-resistant, patient-derived tumor xenografts that mimicked the development of acquired resistance to sorafenib or lenvatinib treatment observed in patients with HCC. RNA sequencing analysis revealed that cholesterol biosynthesis was most commonly enriched in the drug-resistant xenografts. Comparison of the genetic profiles of CD133+ stem cells and CD133- bulk cells from liver regeneration and HCC mouse models showed that the cholesterol pathway was preferentially upregulated in liver CSCs compared with normal liver stem cells. Consistently, SREBP2-mediated cholesterol biosynthesis was crucial for the augmentation of liver CSCs, and loss of SREBP2 conferred sensitivity to tyrosine kinase inhibitors, suggesting a role in regulation of acquired drug resistance in HCC. Similarly, exogenous cholesterol-treated HCC cells showed enhanced cancer stemness abilities and drug resistance. Mechanistically, caspase-3 (CASP3) mediated cleavage of SREBP2 from the endoplasmic reticulum to promote cholesterol biosynthesis, which consequently caused resistance to sorafenib/lenvatinib treatment by driving activation of the sonic hedgehog signaling pathway. Simvastatin, an FDA-approved cholesterol-lowering drug, not only suppressed HCC tumor growth but also sensitized HCC cells to sorafenib. These findings demonstrate that CSC populations in HCC expand via CASP3-dependent, SREBP2-mediated cholesterol biosynthesis in response to tyrosine kinase inhibitor therapy and that targeting cholesterol biosynthesis can overcome acquired drug resistance. SIGNIFICANCE: This study finds that cholesterol biosynthesis supports the expansion of cancer stem cell populations to drive resistance to tyrosine kinase inhibitor therapy in hepatocellular carcinoma, identifying potential therapeutic approaches for improving cancer treatment.

Comment on Cancer Res. 2022 Sep 2;82(17):2980-3001.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies. Elucidating the underlying mechanisms of this disease could provide new therapeutic strategies for treating HCC. Here, we identified a novel role of DEAD-box helicase 24 (DDX24), a member of the DEAD-box protein family, in promoting HCC progression. DDX24 levels were significantly elevated in HCC tissues and were associated with poor prognosis of HCC. Overexpression of DDX24 promoted HCC migration and proliferation in vitro and in vivo, whereas suppression of DDX24 inhibited both functions. Mechanistically, DDX24 bound the mRNA618-624nt of laminin subunit beta 1 (LAMB1) and increased its stability in a manner dependent upon the interaction between nucleolin and the C-terminal region of DDX24. Moreover, regulatory factor X8 (RFX8) was identified as a DDX24 promoter-binding protein that transcriptionally upregulated DDX24 expression. Collectively, these findings demonstrate that the RFX8/DDX24/LAMB1 axis promotes HCC progression, providing potential therapeutic targets for HCC. SIGNIFICANCE: The identification of a tumor-promoting role of DDX24 and the elucidation of the underlying regulatory mechanism provide potential prognostic indicators and therapeutic approaches to help improve the outcome of patients with hepatocellular carcinoma.

Considering the dismal prognosis of castration-resistant prostate cancer (CRPC), it is critical to identify novel therapeutic targets in this disease. Malignant cells have metabolic dependencies distinct from their healthy counterparts, resulting in therapeutic vulnerabilities. Although PTEN and TP53 are the most frequently comutated or codeleted driver genes in lethal CRPC, the metabolic dependencies underlying PTEN/p53 deficiency-driven CRPC for therapeutic intervention remain largely elusive. In this study, PTEN/p53 deficient tumors were determined to be reliant on cholesterol metabolism. Moreover, PTEN/p53 deficiency transcriptionally upregulated squalene epoxidase (SQLE) via activation of sterol regulatory element-binding protein 2 (SREBP2). In addition, PTEN deficiency enhanced the protein stability of SQLE by inhibiting the PI3K/Akt/GSK3β-mediated proteasomal pathway. Consequently, SQLE increased cholesterol biosynthesis to facilitate tumor cell growth and survival. Pharmacologic blockade of SQLE with FR194738 profoundly suppressed the invasive program of CRPC. Collectively, these results demonstrate a synergistic relationship between SQLE and PTEN/p53 deficiency in CRPC development and progression. Therefore, pharmacologic interventions targeting SQLE may hold promise for the treatment of patients with CRPC. SIGNIFICANCE: This study reveals PTEN and p53 deficiency confers a dependence on SQLE-mediated cholesterol metabolism, providing insights for new therapeutic strategies for treating castration-resistant prostate cancer.

Comment on Cancer Res. 2022 Sep 2;82(17):3158-3171.

The mTOR is a master regulator of cell growth that controls cell homeostasis in response to nutrients, growth factors, and other environmental cues. Recent studies have emphasized the importance of lysosomes as a hub for nutrient sensing, especially amino acid sensing by mTORC1. This review highlights recent advances in understanding the amino acid-mTORC1 signaling axis and the role of mTORC1 in cancer.

Metabolic reprogramming is a hallmark of cancer progression. Metabolic activity supports tumorigenesis and tumor progression, allowing cells to uptake essential nutrients from the environment and use the nutrients to maintain viability and support proliferation. The metabolic pathways of malignant cells are altered to accommodate increased demand for energy, reducing equivalents, and biosynthetic precursors. Activated oncogenes coordinate with altered metabolism to control cell-autonomous pathways, which can lead to tumorigenesis when abnormalities accumulate. Clinical and preclinical studies have shown that targeting metabolic features of hematologic malignancies is an appealing therapeutic approach. This review provides a comprehensive overview of the mechanisms of metabolic reprogramming in hematologic malignancies and potential therapeutic strategies to target cancer metabolism.

©2022 American Association for Cancer Research.

Comment in Cancer Res. 2022 Sep 2;82(17):2975-2976.

Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

Single-cell analysis of healthy lung tissue and lung cancer reveals distinct tumor cell populations, including cells with differential immune modulating capacity between smokers and never smokers, which could guide future therapeutic strategies.

Somatic copy-number alterations (CNA) promote cancer, but the underlying driver genes may not be comprehensively identified if only the functions of the encoded proteins are considered. mRNAs can act as competitive endogenous RNAs (ceRNA), which sponge miRNAs to posttranscriptionally regulate gene expression in a protein coding-independent manner. We investigated the contribution of ceRNAs to the oncogenic effects of CNAs. Chromosome 1q gains promoted melanoma progression and metastasis at least in part through overexpression of three mRNAs with ceRNA activity: CEP170, NUCKS1, and ZC3H11A. These ceRNAs enhanced melanoma metastasis by sequestering tumor suppressor miRNAs. Orthogonal genetic assays with miRNA inhibitors and target site blockers, along with rescue experiments, demonstrated that miRNA sequestration is critical for the oncogenic effects of CEP170, NUCKS1, and ZC3H11A mRNAs. Furthermore, chromosome 1q ceRNA-mediated miRNA sequestration alleviated the repression of several prometastatic target genes. This regulatory RNA network was evident in other cancer types, suggesting chromosome 1q ceRNA deregulation as a common driver of cancer progression. Taken together, this work demonstrates that ceRNAs mediate the oncogenicity of somatic CNAs. SIGNIFICANCE: The function of CEP170, NUCKS1, and ZC3H11A mRNAs as competitive endogenous RNAs that sequester tumor suppressor microRNAs underlies the oncogenic activity of chromosome 1q gains.

The X-linked gene DDX3X encodes an RNA helicase that is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis, causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, DDX3X deficiency altered hematopoietic progenitors, early lymphoid development, marginal zone and germinal center B cells, and lymphomagenesis in a sex-dependent manner. Loss of both Ddx3x alleles abrogated MYC-driven lymphomagenesis in females, whereas Ddx3x deletion in males did not affect the formation of B-cell lymphoma in both mouse models. Moreover, tumors that appeared in male mice lacking DDX3X showed upregulated expression of DDX3Y, indicating a critical requirement for DDX3 activity for lymphomagenesis. These data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis. SIGNIFICANCE: The sex-dependent effects of DDX3X deficiency in malignant transformation of B cells and the compensatory role of DDX3Y support inhibition of DDX3 as a treatment strategy for MYC-driven B-cell lymphoma.

Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the in vitro growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin β1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1+ and NEUROD1+ SCLC cells in vitro and suppressed ASCL1+ tumor growth in vivo, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC. SIGNIFICANCE: The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.

Epithelial squamous cell carcinomas (SCC) most commonly originate in the skin, where they display disruptions in the normally tightly regulated homeostatic balance between keratinocyte proliferation and terminal differentiation. We performed a transcriptome-wide screen for genes of unknown function that possess inverse expression patterns in differentiating keratinocytes compared with cutaneous SCC (cSCC), leading to the identification of MAB21L4 (C2ORF54) as an enforcer of terminal differentiation that suppresses carcinogenesis. Loss of MAB21L4 in human cSCC organoids increased expression of RET to enable malignant progression. In addition to transcriptional upregulation of RET, deletion of MAB21L4 preempted recruitment of the CacyBP-Siah1 E3 ligase complex to RET and reduced its ubiquitylation. In SCC organoids and in vivo tumor models, genetic disruption of RET or selective inhibition of RET with BLU-667 (pralsetinib) suppressed SCC growth while inducing concomitant differentiation. Overall, loss of MAB21L4 early during SCC development blocks differentiation by increasing RET expression. These results suggest that targeting RET activation is a potential therapeutic strategy for treating SCC. SIGNIFICANCE: Downregulation of RET mediated by MAB21L4-CacyBP interaction is required to induce epidermal differentiation and suppress carcinogenesis, suggesting RET inhibition as a potential therapeutic approach in squamous cell carcinoma.

Clinical studies have shown that subsets of patients with cancer achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain helicase DNA binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the coactivator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. SIGNIFICANCE: CHD1 plays a critical role in controlling AURKA activation and promoting Aurora kinase inhibitor sensitivity, providing a potential clinical biomarker to guide cancer treatment.

Tumor-associated macrophages (TAM) play a detrimental role in triple-negative breast cancer (TNBC). In-depth analysis of TAM characteristics and interactions with stromal cells, such as cancer-associated fibroblast (CAF), could provide important biological and therapeutic insights. Here we identify at the single-cell level a monocyte-derived STAB1+TREM2high lipid-associated macrophage (LAM) subpopulation with immune suppressive capacities that is expanded in patients resistant to immune checkpoint blockade (ICB). Genetic depletion of this LAM subset in mice suppressed TNBC tumor growth. Flow cytometry and bulk RNA sequencing data demonstrated that coculture with TNBC-derived CAFs led to reprogramming of blood monocytes towards immune suppressive STAB1+TREM2high LAMs, which inhibit T-cell activation and proliferation. Cell-to-cell interaction modeling and assays in vitro demonstrated the role of the inflammatory CXCL12-CXCR4 axis in CAF-myeloid cell cross-talk and recruitment of monocytes in tumor sites. Altogether, these data suggest an inflammation model whereby monocytes recruited to the tumor via the CAF-driven CXCL12-CXCR4 axis acquire protumorigenic LAM capacities to support an immunosuppressive microenvironment. SIGNIFICANCE: This work identifies a novel lipid-associated macrophage subpopulation with immune suppressive functions, offering new leads for therapeutic interventions in triple-negative breast cancer.

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. SIGNIFICANCE: β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been previously implicated in activities consistent with tumor suppression. Here we generated Gls2 knockout (KO) mice that develop late-occurring B-cell lymphomas and hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors than seen in wild-type (WT) mice. GLS2 has been shown to promote ferroptosis, a form of cell death characterized by iron-dependent accumulation of lipid peroxides. In line with this, GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, but not GLS1, increased lipid reactive oxygen species (ROS) production by facilitating the conversion of glutamate to α-ketoglutarate (αKG), thereby promoting ferroptosis. Ectopic expression of WT GLS2 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; this effect was nullified by either expressing a catalytically inactive form of GLS2 or by blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function. SIGNIFICANCE: This study demonstrates that the key regulator of glutaminolysis, GLS2, can limit HCC in vivo by promoting ferroptosis through αKG-dependent lipid ROS, which in turn might lay the foundation for a novel therapeutic approach.

Hepatocellular carcinoma (HCC) is one of the primary liver malignancies with a poor prognosis. Glutamic-oxaloacetic transaminase 2 (GOT2) is a highly tissue-specific gene in the liver, but the roles GOT2 plays in the progression of HCC remain unclear. Here, we report that GOT2 is downregulated in HCC tumor tissues and that low expression of GOT2 is associated with advanced progression and poor prognosis. In HCC cells, knockdown of GOT2 promoted proliferation, migration, and invasion. In mouse models of HCC, loss of GOT2 promoted tumor growth as well as hematogenous and intrahepatic metastasis. Mechanistically, silencing of GOT2 enhanced glutaminolysis, nucleotide synthesis, and glutathione synthesis by reprogramming glutamine metabolism to support the cellular antioxidant system, which activated the PI3K/AKT/mTOR pathway to contribute to HCC progression. Furthermore, HCC with low expression of GOT2 was highly dependent on glutamine metabolism and sensitive to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, GOT2 is involved in glutamine metabolic reprogramming to promote HCC progression and may serve as a therapeutic and diagnostic target for HCC. SIGNIFICANCE: Altered glutamine metabolism induced by GOT2 loss supports HCC growth and metastasis but confers a targetable vulnerability to glutaminase inhibitors.

Glioblastoma (GBM) is among the most aggressive human cancers. Although oncolytic virus (OV) therapy has been proposed as a potential approach to treat GBM, it frequently fails because GBM cells are usually nonpermissive to OV. Here, we describe a dual-step drug screen for identifying chemical enhancers of OV in GBM. From a high-throughput screen of 1416 FDA-approved drugs, an inhibitor of CDK4/6 was identified as the top enhancer, selectively increasing potency of two OV strains, VSVΔ51 and Zika virus. Mechanistically, CDK4/6 inhibition promoted autophagic degradation of MAVS, resulting in impaired antiviral responses and enhanced tumor-selective replication of VSVΔ51 in vitro and in vivo. CDK4/6 inhibition cooperated with VSVΔ51 to induce severe DNA damage stress and amplify oncolysis. In GBM xenograft models, combined treatment with CDK4/6 inhibitor and VSVΔ51 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. Further investigation revealed that CDK4/6 inhibitor and VSVΔ51 synergistically induced immunogenic cell death and boosted antitumor immunity. Together, this study features a promising approach of treating aggressive GBM through the combination of CDK4/6 inhibitor with OV. SIGNIFICANCE: This study proposes inhibition of cyclin-dependent kinases as a clinically translatable combinatorial strategy to enhance the efficacy of oncolytic virotherapy in GBM.

The BRCA1-PALB2-BRCA2 axis plays essential roles in the cellular response to DNA double-strand breaks (DSB), maintenance of genome integrity, and suppression of cancer development. Upon DNA damage, BRCA1 is recruited to DSBs, where it facilitates end resection and recruits PALB2 and its associated BRCA2 to load the central recombination enzyme RAD51 to initiate homologous recombination (HR) repair. In recent years, several BRCA1-independent mechanisms of PALB2 recruitment have also been reported. Collectively, these available data illustrate a series of hierarchical, context-dependent, and cooperating mechanisms of PALB2 recruitment that is critical for HR and therapy response either in the presence or absence of BRCA1. Here, we review these BRCA1-dependent and independent mechanisms and their importance in DSB repair, cancer development, and therapy. As BRCA1-mutant cancer cells regain HR function, for which PALB2 is generally required, and become resistant to targeted therapies, such as PARP inhibitors, targeting BRCA1-independent mechanisms of PALB2 recruitment represents a potential new avenue to improve treatment of BRCA1-mutant tumors.

Cancer immunotherapy restores or enhances the effector function of T cells in the tumor microenvironment, but the efficacy of immunotherapy has been hindered by therapeutic resistance. Here, we identify the proto-oncogene serine/threonine protein kinase PIM2 as a novel negative feedback regulator of IFNγ-elicited tumor inflammation, thus endowing cancer cells with aggressive features. Mechanistically, IL1β derived from IFNγ-polarized tumor macrophages triggered PIM2 expression in cancer cells via the p38 MAPK/Erk and NF-κB signaling pathways. PIM2+ cancer cells generated by proinflammatory macrophages acquired the capability to survive, metastasize, and resist T-cell cytotoxicity and immunotherapy. A therapeutic strategy combining immune checkpoint blockade (ICB) with IL1β blockade or PIM2 kinase inhibition in vivo effectively and successfully elicited tumor regression. These results provide insight into the regulatory and functional features of PIM2+ tumors and suggest that strategies to influence the functional activities of inflammatory cells or PIM2 kinase may improve the efficacy of immunotherapy. SIGNIFICANCE: Cross-talk between T cells and macrophages regulates cancer cell PIM2 expression to promote cancer aggressiveness, revealing translational approaches to improve response to ICB in hepatocellular carcinoma.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development. SIGNIFICANCE: This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.

Comment on Cancer Res. 2022 Sep 16;82(18):3275-3290.

Women who have had breast cancer in the past are at increased risk of developing a second primary cancer (SPC), including second primary breast cancer (SPBC) or a second primary non-breast cancer (SPNBC). In the Multiethnic Cohort (MEC) Study, we conducted a prospective cohort analysis in 3,223 female breast cancer survivors from five racial/ethnic populations (White, African American, Japanese American, Latino, and Native Hawaiian) to assess the association of rare pathogenic variants (PV) in 37 known cancer predisposition genes with risk of SPC. A total of 719 (22.3%) women developed SPC, of which, 323 (10.0%) were SPBC. Germline PVs in BRCA1 (HR, 2.28; 95% CI, 1.11-4.65) and ERCC2 (HR, 3.51; 95% CI, 1.29-9.54) were significantly enriched in women with SPC. In the subtype analysis for SPBC, a significant association of ERCC2 PVs (HR, 5.09; 95% CI, 1.58-16.4) and a suggestive association of BRCA2 PVs (HR, 2.24; 95% CI, 0.91-5.55) were observed. There was also a higher risk of SPNBC in carriers of BRCA1 PVs (HR, 2.98; 95% CI, 1.21-7.36). These results provide evidence that germline PVs in BRCA1, BRCA2, and ERCC2 contribute to the development of SPC in breast cancer survivors. These findings also suggest that compromised DNA repair mechanisms could be a predisposition factor for SPC in patients with breast cancer, supporting the need for closer monitoring of SPC in women carrying PVs in these genes. SIGNIFICANCE: This multiethnic study links germline pathogenic variants in BRCA1, BRCA2, and ERCC2 to the development of second primary cancer in breast cancer survivors, providing biological insights and biomarkers to guide patient monitoring.

A subset of cancers across multiple histologies with predominantly poor outcomes use the alternative lengthening of telomeres (ALT) mechanism to maintain telomere length, which can be identified with robust biomarkers. ALT has been reported to be prevalent in high-risk neuroblastoma and certain sarcomas, and ALT cancers are a major clinical challenge that lack targeted therapeutic approaches. Here, we found ALT in a variety of pediatric and adult cancer histologies, including carcinomas. Patient-derived ALT cancer cell lines from neuroblastomas, sarcomas, and carcinomas were hypersensitive to the p53 reactivator eprenetapopt (APR-246) relative to telomerase-positive (TA+) models. Constitutive telomere damage signaling in ALT cells activated ataxia-telangiectasia mutated (ATM) kinase to phosphorylate p53, which resulted in selective ALT sensitivity to APR-246. Treatment with APR-246 combined with irinotecan achieved complete responses in mice xenografted with ALT neuroblastoma, rhabdomyosarcoma, and breast cancer and delayed tumor growth in ALT colon cancer xenografts, while the combination had limited efficacy in TA+ tumor models. A large number of adult and pediatric cancers present with the ALT phenotype, which confers a uniquely high sensitivity to reactivation of p53. These data support clinical evaluation of a combinatorial approach using APR-246 and irinotecan in ALT patients with cancer. SIGNIFICANCE: This work demonstrates that constitutive activation of ATM in chemotherapy-refractory ALT cancer cells renders them hypersensitive to reactivation of p53 function by APR-246, indicating a potential strategy to overcome therapeutic resistance.

The mTOR is a key regulator of cell growth that integrates growth factor signaling and nutrient availability and is a downstream effector of oncogenic receptor tyrosine kinases (RTK) and PI3K/Akt signaling. Thus, activating mTOR mutations would be expected to enhance growth in many tumor types. However, tumor sequencing data have shown that mTOR mutations are enriched only in renal clear cell carcinoma, a clinically hypervascular tumor unlikely to be constrained by nutrient availability. To further define this cancer-type-specific restriction, we studied activating mutations in mTOR. All mTOR mutants tested enhanced growth in a cell-type agnostic manner under nutrient-replete conditions but were detrimental to cell survival in nutrient-poor conditions. Consistently, analysis of tumor data demonstrated that oncogenic mutations in the nutrient-sensing arm of the mTOR pathway display a similar phenotype and were exceedingly rare in human cancers of all types. Together, these data suggest that maintaining the ability to turn off mTOR signaling in response to changing nutrient availability is retained in most naturally occurring tumors. SIGNIFICANCE: This study suggests that cells need to inactivate mTOR to survive nutrient stress, which could explain the rarity of mTOR mutations and the limited clinical activity of mTOR inhibitors in cancer.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC. SIGNIFICANCE: Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

The Fanconi anemia (FA) pathway is essential for repairing DNA interstrand crosslinks (ICL). ICLs induce stalled DNA replication forks and trigger activation of the FA pathway by promoting recruitment of the FANCM/FAAP24/MHF complex to ICL sites. Given that stalled replication forks are proximal to ICL sites, fork-associated proteins may coordinate with FA factors to rapidly sense ICLs for activation of FA signaling. Here we report that And-1, a replisome protein, is critical for activation of the FA pathway by sensing ICL-stalled forks and recruiting the FANCM/FAAP24 complex to ICLs. In response to ICLs, And-1 rapidly accumulated at ICL-stalled forks in a manner dependent on ataxia telangiectasia and Rad3-related protein-induced phosphorylation at T826. And-1 phosphorylation triggered an intramolecular change that promoted the interaction of And-1 with FANCM/FAAP24, resulting in recruitment of the FANCM/FAAP24 complex to ICLs. Furthermore, p-T826 And-1 was elevated in cisplatin-resistant ovarian cancer cells, and activated And-1 contributed to cisplatin resistance. Collectively, these studies elucidate a mechanism by which And-1 regulates FA signaling and identify And-1 as a potential target for developing therapeutic approaches to treat platinum-resistant ovarian cancer. SIGNIFICANCE: This work shows that phosphorylation of And-1 by ATR activates Fanconi anemia signaling at interstrand crosslink-stalled replication forks by recruiting the FANCM/FAAP24 complex, revealing And-1 as a potential therapeutic target in cancer.

Comment in Cancer Res. 2022 Sep 16;82(18):3198-3200.

Triple-negative breast cancer (TNBC) is persistently refractory to therapy, and methods to improve targeting and evaluation of responses to therapy in this disease are needed. Here, we integrate quantitative MRI data with biologically based mathematical modeling to accurately predict the response of TNBC to neoadjuvant systemic therapy (NAST) on an individual basis. Specifically, 56 patients with TNBC enrolled in the ARTEMIS trial (NCT02276443) underwent standard-of-care doxorubicin/cyclophosphamide (A/C) and then paclitaxel for NAST, where dynamic contrast-enhanced MRI and diffusion-weighted MRI were acquired before treatment and after two and four cycles of A/C. A biologically based model was established to characterize tumor cell movement, proliferation, and treatment-induced cell death. Two evaluation frameworks were investigated using: (i) images acquired before and after two cycles of A/C for calibration and predicting tumor status after A/C, and (ii) images acquired before, after two cycles, and after four cycles of A/C for calibration and predicting response following NAST. For Framework 1, the concordance correlation coefficients between the predicted and measured patient-specific, post-A/C changes in tumor cellularity and volume were 0.95 and 0.94, respectively. For Framework 2, the biologically based model achieved an area under the receiver operator characteristic curve of 0.89 (sensitivity/specificity = 0.72/0.95) for differentiating pathological complete response (pCR) from non-pCR, which is statistically superior (P < 0.05) to the value of 0.78 (sensitivity/specificity = 0.72/0.79) achieved by tumor volume measured after four cycles of A/C. Overall, this model successfully captured patient-specific, spatiotemporal dynamics of TNBC response to NAST, providing highly accurate predictions of NAST response. SIGNIFICANCE: Integrating MRI data with biologically based mathematical modeling successfully predicts breast cancer response to chemotherapy, suggesting digital twins could facilitate a paradigm shift from simply assessing response to predicting and optimizing therapeutic efficacy.