KRAS-driven Lung Cancer Research Articles

Inhibition of lung tumorigenesis by transient reprogramming in cancer cells

Oncogenic transformation and Oct4, Sox2, Klf4 and c-Myc (OSKM)-mediated induction of pluripotency are two independent and incompatible cellular fates. While continuous expression of OSKM can convert normal somatic cells into teratogenic pluripotent cells, it remains speculative what is the impact of transient OSKM expression in cancer cells. Here, we find that OSKM expression limits the growth of transformed lung cells by inducing apoptosis and senescence. We identify Oct4 and Klf4 as the main individual reprogramming factors responsible for this effect. Mechanistically, the induction of cell cycle inhibitor p21 downstream of the reprogramming factors acts as mediator of cell death and senescence. Using a variety of in vivo systems, including allografts, orthotopic transplantation and KRAS-driven lung cancer mouse models, we demonstrate that transient reprogramming by OSKM expression in cancer cells impairs tumor growth and reduces tumor burden. Altogether, our results show that the induction of transient reprogramming in cancer cells is antitumorigenic opening novel potential therapeutic avenues in oncology.

Tenascin-C in the early lung cancer tumor microenvironment promotes progression through integrin αvβ1 and FAK.

Pre-cancerous lung lesions are commonly initiated by activating mutations in the RAS pathway, but do not transition to lung adenocarcinomas (LUAD) without additional oncogenic signals. Here, we show that expression of the extracellular matrix protein Tenascin-C (TNC) is increased in and promotes the earliest stages of LUAD development in oncogenic KRAS-driven lung cancer mouse models and in human LUAD. TNC is initially expressed by fibroblasts and its expression extends to tumor cells as the tumor becomes invasive. Genetic deletion of TNC in the mouse models reduces early tumor burden and high-grade pathology and diminishes tumor cell proliferation, invasion, and focal adhesion kinase (FAK) activity. TNC stimulates cultured LUAD tumor cell proliferation and migration through engagement of αv-containing integrins and subsequent FAK activation. Intringuingly, lung injury causes sustained TNC accumulation in mouse lungs, suggesting injury can induce additional TNC signaling for early tumor cell transition to invasive LUAD. Biospecimens from patients with stage I/II LUAD show TNC in regions of FAK activation and an association of TNC with tumor recurrence after primary tumor resection. These results suggest that exogenous insults that elevate TNC in the lung parenchyma interact with tumor-initiating mutations to drive early LUAD progression and local recurrence.

Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1-Mutant Lung Cancer.

The acquisition of invasive properties is a prerequisite for tumor progression and metastasis. Molecular subtypes of KRAS-driven lung cancer exhibit distinct modes of invasion that contribute to unique growth properties and therapeutic susceptibilities. Despite this, preclinical strategies designed to exploit growth within the context of invasion are lacking. To address this, we designed an experimental system to screen for targetable signaling pathways linked to active early 3D invasion phenotypes in different molecular subtypes of KRAS-driven lung adenocarcinoma. Combined live-cell imaging of human bronchial epithelial cells in a 3D invasion matrix and transcriptomic profiling identified mutant LKB1-specific upregulation of BMP6. LKB1 loss increased BMP6 signaling, which induced the canonical iron regulatory hormone hepcidin. Intact LKB1 was necessary to maintain BMP6 signaling homeostasis and restrict ALK2/BMP6-fueled growth. Preclinical studies in a Kras/Lkb1-mutant syngeneic mouse model and in a xenograft model showed potent growth suppression by inhibiting the ALK2/BMP6 signaling axis with single-agent inhibitors that are currently in clinical trials. Lastly, BMP6 expression was elevated in tumors of patients with LKB1-mutant early-stage lung cancer. These results are consistent with those of a model in which LKB1 acts as a "brake" to iron-regulated growth and suggest that ALK2 inhibition can be used for patients with LKB1-mutant tumors. Significance: Three-dimensional invasion-linked gene expression analysis reveals a therapeutic vulnerability to inhibition of ALK2/BMP6 signaling in LKB1-mutant lung cancer that can be rapidly translated to the clinic.

Glucose-6-phosphate dehydrogenase maintains redox homeostasis and biosynthesis in LKB1-deficient KRAS-driven lung cancer

Cancer cells depend on nicotinamide adenine dinucleotide phosphate (NADPH) to combat oxidative stress and support reductive biosynthesis. One major NADPH production route is the oxidative pentose phosphate pathway (committed step: glucose-6-phosphate dehydrogenase, G6PD). Alternatives exist and can compensate in some tumors. Here, using genetically-engineered lung cancer mouse models, we show that G6PD ablation significantly suppresses KrasG12D/+;Lkb1-/- (KL) but not KrasG12D/+;P53-/- (KP) lung tumorigenesis. In vivo isotope tracing and metabolomics reveal that G6PD ablation significantly impairs NADPH generation, redox balance, and de novo lipogenesis in KL but not KP lung tumors. Mechanistically, in KL tumors, G6PD ablation activates p53, suppressing tumor growth. As tumors progress, G6PD-deficient KL tumors increase an alternative NADPH source from serine-driven one carbon metabolism, rendering associated tumor-derived cell lines sensitive to serine/glycine depletion. Thus, oncogenic driver mutations determine lung cancer dependence on G6PD, whose targeting is a potential therapeutic strategy for tumors harboring KRAS and LKB1 co-mutations.

Cytosolic DNA sensor AIM2 promotes KRAS-driven lung cancer independent of inflammasomes.

Constitutively active KRAS mutations are among the major drivers of lung cancer, yet the identity of molecular co-operators of oncogenic KRAS in the lung remains ill-defined. The innate immune cytosolic DNA sensor and pattern recognition receptor (PRR) Absent-in-melanoma 2 (AIM2) is best known for its assembly of multiprotein inflammasome complexes and promoting an inflammatory response. Here, we define a role for AIM2, independent of inflammasomes, in KRAS-addicted lung adenocarcinoma (LAC). In genetically defined and experimentally induced (nicotine-derived nitrosamine ketone; NNK) LAC mouse models harboring the KrasG12D driver mutation, AIM2 was highly upregulated compared with other cytosolic DNA sensors and inflammasome-associated PRRs. Genetic ablation of AIM2 in KrasG12D and NNK-induced LAC mouse models significantly reduced tumor growth, coincident with reduced cellular proliferation in the lung. Bone marrow chimeras suggest a requirement for AIM2 in KrasG12D-driven LAC in both hematopoietic (immune) and non-hematopoietic (epithelial) cellular compartments, which is supported by upregulated AIM2 expression in immune and epithelial cells of mutant KRAS lung tissues. Notably, protection against LAC in AIM2-deficient mice is associated with unaltered protein levels of mature Caspase-1 and IL-1β inflammasome effectors. Moreover, genetic ablation of the key inflammasome adapter, ASC, did not suppress KrasG12D-driven LAC. In support of these invivo findings, AIM2, but not mature Caspase-1, was upregulated in human LAC patient tumor biopsies. Collectively, our findings reveal that endogenous AIM2 plays a tumor-promoting role, independent of inflammasomes, in mutant KRAS-addicted LAC, and suggest innate immune DNA sensing may provide an avenue to explore new therapeutic strategies in lung cancer.

Abstract LB077: CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models

Abstract Background: KRAS mutations are highly prevalent oncogenic drivers in human cancers and KRASG12C is the most frequent mutations found in KRAS-mutant non-small lung cancer (NSCLC). It is reported that tumor associated macrophages (TAMs) are enriched in KRAS-driven transgenic lung cancer models. Depletion of macrophage results in a significant decrease in tumor burden and increased survival. Sotorasib (AMG510), the first KRASG12C inhibitor approved by FDA, has been found to modulate tumor immunity, impacting the recruitment of macrophages. CSF-1R signaling is critical for macrophage proliferation and survival. CSF-1R inhibition depletes TAMs and reprograms tumor microenvironment (TME), resulting in enhancement of anti-tumor immunity. Therefore, the combination of KRASG12C and CSF-1R inhibitors may synergize to enhance anti-tumor efficacy. Pimicotinib (ABSK021) is a potential best-in-class small molecule inhibitor of CSF-1R in clinical development of multiple indications. Here, we conducted in vivo experiments to explore the combined effects of KRASG12C and CSF-1R inhibitors. Methods: In vivo efficacy studies were performed to evaluate the combined treatment of KRASG12C inhibitors (AMG510 and ABSK071, a next-generation inhibitor targeting KRASG12C with better inhibitory activity) and CSF-1R inhibitor (ABSK021) in NCI-H2122 PBMC humanized mice and LLC syngeneic mice. RNA sequencing was employed to investigate the mechanism of action underlying the synergistic anti-tumor effect. Immunohistochemistry (IHC) was utilized to examine the changes of immune cell populations in TME. Results: The combinatory regimen of ABSK021 and KRASG12C inhibitors showed improved anti-tumor effects than each single agent alone in both mouse NSCLC models. Bioinformatics and follow-up analysis revealed a reduction in immune suppressive TAMs in tumors treated with ABSK021 alone or in combination with KRASG12C inhibitors. Furthermore, modulation of TME related to anti-tumor immunity, such as activated CD4 T, CD8 T and NK cells, was increased, and pro-tumor immunity, such as MDSC, regulatory T cells, was decreased in combination group. IHC results confirmed that ABSK021 addition led to macrophage depletion and more CD8 T cell infiltration in TME. Conclusions: For the first time, we demonstrate that the combined inhibition of KRASG12C and CSF-1R leads to superior therapeutic efficacy in preclinical NSCLC mouse models. This results suggests a potential novel therapeutic regimen that could yield improved clinical benefit to patients. Citation Format: Nannan Zhang, Bin Shen, Shenyan Liu, Cheng Dai, Haiyan Ying, Fushen Guo, Zhixuan Zhu, Wan Shi, Min Tu, Jie Wang, Jie Zhang, Manqi Liu, Qi Zhang, Zhui Chen. CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB077.

Abstract 7470: Leukemia inhibitory factor promotes tumor progression in KRAS-driven lung cancer

Abstract Leukemia inhibitory factor (LIF) displays complex effects in different types of cancer but its role in lung cancer remains unknown. Since KRAS mutation is an initial validated oncogenic event in near 30% of non-small cell lung cancer (PanCancer Atlas), we were interested in studying how LIF would influence tumor initiation and progression in KRAS-driven lung cancer.To properly study the roles of LIF throughout KRAS-driven tumorigenesis, we generated a novel genetically engineered mouse model (GEMM), where expression of LIF can be conditionally knocked out by Cre recombinase. Conditional deletion of the Lif allele in oncogenic KRAS-induced lung tumors is achieved by crossing Lif flox/flox mice with KrasLSL-G12D/+, Trp53flox/flox (KP) mouse model. In our lung cancer model, induction of oncogenic KRAS and depletion of LIF is attained through intratracheal infection of adeno-Cre virus.When our Lif flox/floxKP (LKP) and KP mice were infected by adeno-Cre virus, they developed lung tumors at reasonable frequencies within 4-8 months. We generated cell lines from tumors and used ELISA and RT-qPCR analysis to validate the efficiency of the knockout. While control KP tumor cell lines (n=3) showed Lif expression and LIF protein secretion, cell lines from LKP tumors (n=3) presented with undetectable Lif expression and no LIF secretion validating the successful deletion of Lif and functionality of our new mouse model. We generated H&E slides from the tumors and with the help of our pathologist we confirmed the subtype of lung adenocarcinoma. The effects of LIF on cell lines proliferation were evaluated in vitro and we found that LKP cell lines grow significantly slower than the KP lines (p<0.05).Mice in both groups were monitored for their overall survival (OS) rate. LKP mice (n=5) tend to have an increased median overall survival at 176 days when compared to control KP mice (n=7) at 152 days (p=0.07). To estimate the tumor initiation and progression, mice in each group were sacrificed at early endpoints (10- and 15-weeks post adeno-cre infection) and ratios of lung weight/mouse weight were measured as an indicator of tumor burden. At 10 weeks, LKP mice (n=5) presented with significantly smaller ratios with an average of 0.014 compared to KP animals (n=4) with an average of 0.019 (p=0.027).We found that LIF tends to shorten overall survival and augments tumor burden in lung cancer. These data suggest that LIF has a pro-tumorigenic role in KRAS-driven lung cancer proposing this cytokine as a potential prognostic biomarker and its depletion as a promising therapeutic target. Citation Format: Imene Boukhalfa Hanafi, Lisa Miller-Phillips, Ons Zakraoui, Kieren D. Marini, Sarah Umetsu, Man-Tzu Wang, Eric A. Collisson. Leukemia inhibitory factor promotes tumor progression in KRAS-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7470.

Abstract 3314: Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors

Abstract Menin is an epigenetic protein that drives oncogenic function through transcriptional control directed by its various cofactors such as MLL1 (KMT2A) and MYC. BMF-219 is a covalent menin inhibitor currently under investigation in a dose-finding study evaluating safety and tolerability in adults with KRAS-driven Non-Small Cell Lung Cancer (NSCLC), Pancreatic Adenocarcinoma (PDAC), or Colorectal Cancer (CRC) (NCT NCT05631574). We previously showed that BMF-219 induces effective killing of solid tumor cells irrespective of their KRAS mutation subtype (AACR 2022, abstract 2665) and perturbs genomic binding of MYC and its binding partner MAX, in acute leukemia cells (ASH Abstract 2021). To probe into the underlying mechanism and the potential pathways engaged, BMF-219-induced effects were evaluated in CRC, PDAC and NSCLC cell lines. Cells treated with relevant concentrations of BMF-219 versus vehicle were assessed for menin and MYC protein levels, along with treatment effects on cell cycle and induction of apoptosis. In addition, bulk RNA-seq analysis was performed on the cell lines treated with BMF-219 in a time course study. BMF-219 induces a dose-dependent decrease in menin protein levels in the solid tumor cell lines tested following 24 hours treatment, with the least impact observed in a KRAS WT cell line. The effect of BMF-219 on c-MYC protein levels were variable and highly cell-type dependent, with the CRC metastatic tumor cell line SW620 exhibiting the highest reduction. Differential expression (DE) analysis of bulk RNA-seq data comparing BMF-219 treated versus vehicle treated cells, identified significant alterations in transcript abundance at 6- and 24-hours post treatment. The magnitude of change in the transcript abundance showed a dose-dependent response without significant alteration of a panel of housekeeping genes. GSEA analysis identified pathways associated with cell cycle, cell division and MYC-regulated were significantly differentially expressed across multiple cell lines. These data suggest similar molecular pathways are altered by BMF-219 across KRAS-mutated solid tumor cells. Additional insights into key pathways and modulators will be reported. Collectively, these data suggest that the functional pathway alterations induced by BMF-219 in solid tumor cells are likely pan-KRAS and are similar across different solid tumor indications. This provides further support for advancing the clinical investigation of BMF-219 in KRAS-driven solid tumors. Citation Format: Amin A. Momin, Tripta Rughwani, Nida Tanataweethum, Kasia Mordec, Raechel Lin, Megan Chin, Daniel Lu, Priyanka Somanath, Mini Balakrishnan, Thomas Butler. Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3314.

Abstract 726: The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis

Abstract Immunotherapy is rarely effective in breast cancer and most current therapeutic options fail to target the stromal compartment. We have developed a new small molecule, MSU42011, that induces tumor regression in experimental models of Kras-driven lung cancer and HER2+ breast cancer. This tumor regression is dependent on a functional immune system. Here, we dissected the tumor stroma, revealing a CD4 and IL12a dependency for the anti-tumor activity of MSU42011. Cancer immunotherapy responses are mostly driven by CD8 T cells. Using a murine HER2+ tumor model, anti-CD8 antibodies (200ug/mouse) were injected 24 hours before treatment with MSU42011 (100MG/KG in power diet) was started. Mice were maintained on diet for 10 days. Anti-CD8 antibodies were also administered on days 3, 7 and 10. The combination did not affect the inhibition of tumor growth by MSU42011, suggesting that its anti-tumor efficacy is not dependent on CD8 T cells. In contrast, tumors treated with MSU42011 and anti-CD4 antibodies grew (p=0.0016 versus MSU42011 alone) at the same rate as tumors treated with control diet (p=0.0139 versus MS42011). Furthermore, anti-CD4 reversed inhibition of tumor growth by MSU42011 in a carcinogen-induced lung tumor model. Interestingly, MSU42011 increased the levels of IL12 by 4x (p=0.01) and IFN by 8x (p=0.03) compared to the controls. These data suggest that MSU42011 increased Th1 CD4 T cell polarization; the increase in IL12 was lost following CD4 T cell depletion. Following depletion of IL12a with antibodies (loading dose of 1 mg, followed by 0.5mg per mouse IP), tumors treated with the combination of MSU42011 and anti-IL12a antibodies grew at the same rate as the controls. T cell engagement with MSU42011 suggests that this small molecule is a good candidate for combination with immunotherapy. HER2+ MMTV-Neu mice with established tumors were treated with the combination of MSU42011 and anti-PD1 antibodies. Treatment with MSU42011 at 100mg/Kg of diet for 10 days, followed by anti-PD1 antibodies (200ug) twice weekly failed to increase survival. In contrast, 2 doses of anti-PD1 antibodies (300ug), followed by treatment with MSU42011 increased survival by 10 days compared to controls. The tumors in the MSU42011+anti-PD1 group initially grew rapidly, with increased redness and heat, suggesting an enhanced inflammatory response. When anti-PD1 antibodies were given at a lower loading dose (200ug and then 100ug twice weekly) followed by a higher dose of MSU42011 (300 mg/Kg of diet), survival markedly increased (p=0.052). These findings demonstrate that activation of RXR in the stroma can be used to increase the effectiveness of immunotherapy. Citation Format: Ana Sofia Leal, Karen T. Liby. The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 726.

Abstract A177: In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers

Abstract Lung adenocarcinoma (LUAD) is a complex cancer driven by diverse combinations of oncogenic and tumor suppressive events. The TBX2 subfamily genes, including TBX2, TBX3, TBX4, and TBX5, are known to play a crucial role in lung development as master transcriptional regulators. Our previous clinical and in vitro studies found that their expression is significantly suppressed in LUADs, suggesting a potential tumor suppressor role through modulating tumor methylation patterns in early tumorigenesis. However, the role of TBX2 subfamily genes in other cancer types is paradoxical, with both oncogenic and tumor suppressive effects being reported. Herein, we employed the Tumor Barcoding with Ultradeep Sequencing (Tuba-seq) and CRISPR/Cas9-mediated genome editing technologies to investigate the putative tumor suppressive role of TBX2 genes functionally and quantitatively and their downstream targets in genetically engineered mouse models of LUAD. Lung tumors were initiated by intratracheal intubation with pooled lentiviral-Cre vectors targeting each gene independently with two different sgRNAs per gene. To enable the unique identification of each tumor and its corresponding sgRNA, we utilized Lenti-sgRNA/Cre vectors harboring barcodes specific to each targeted gene. Initial assessment of tumor burden was carried out through fluorescence microscopy, lung weight measurements, and histological analysis. To simultaneously determine the number of neoplastic cells corresponding to each gene knockout, the barcoded region was amplified from genomic DNA extracted from bulk tumor-bearing lung samples and sequenced. Quantification of tumor sizes was carried at 6 and 20 weeks after tumor initiation. Hyperplasia, adenomas, and/or early adenocarcinomas within the lungs were detected among different genetic backgrounds and throughout the study time intervals. Specifically, the inactivation of Tbx2, Tbx3, Tbx4, Tbx5, and Cdh2, increased tumor initiation and growth in the oncogene-negative mouse model (n=8). However, strikingly, inactivation of Tbx3, Tbx4, and Cdh2 suppressed tumorigenesis in a KRAS-driven genetic background while Tbx2, Tbx5, and Tnfaip3, consistently exhibited tumor suppressive effects (n=9). Further investigation is needed to understand these newly discovered interactions between the TBX2 subfamily critical developmental pathway and Ras signaling in modulating lung cancer progression. These insights emphasize the importance of considering tumor suppressor phenotypic heterogeneity and their context-dependent roles when developing targeted therapeutic approaches for lung adenocarcinoma and other cancers. Citation Format: Athar Khalil, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher Mcfarland. In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A177.

Abstract LB_B21: Extracellular RNA signatures of mutant KRAS-driven lung cancer

Abstract RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Using both affinity- and nanofiltration-based EV isolation approaches, we show that specific TE RNAs, lncRNAs, and mRNAs, some of which are prognostic for lung adenocarcinoma (LUAD) patient survival, are preferentially released in extracellular vesicles (EVs). Moreover, we find that inhibition of oncogenic KRAS signaling broadly reprograms the noncoding transcriptome, as evidenced by a substantial increase in TE RNA secretion. Mutant KRAS inhibition also increases the abundance of secreted lncRNAs and retained intron-containing transcripts, while decreasing the mRNA content of EVs. Notably, oncogenic KRAS signaling is required for the secretion of mRNAs from a set of 20 genes that are significantly associated with unfavorable clinical outcomes in LUAD. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway. Our study also suggests that both coding and noncoding RNAs that are secreted in EVs may serve as mutant KRAS-specific signatures for diagnosing lung cancer via minimally invasive RNA liquid biopsy technology, which we are currently developing as a companion diagnostic for mutant KRAS-targeting therapies. Citation Format: Sree Lakshmi Velandi Maroli, Roman E Reggiardo, Reem Khojah, Daniel H Kim. Extracellular RNA signatures of mutant KRAS-driven lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B21.

Polymeric nano-formulation of spectrum selective RTK inhibitor strengthens anti-cancer effects via immune remodeling by endoplasmic reticulum stress-modulating mitochondrial metabolism

The tumor microenvironment (TME), where immunosuppressive cells such as tumor-associated macrophages (TAMs) proliferate, is the main cause of resistance to antineoplastic treatment for KRAS-driven lung cancer. In this study, we synthesized polymer-based nanoparticles composed of a 16 nm-sized Au core and the amphiphile, poly-(styrene-alt-maleic acid) (PSMA), via a hydrothermal procedure for carrying the multi-receptor tyrosine kinase inhibitor, sitravatinib (Sit), in a new nanodrug (Au@PSMA-Sit). Au@PSMA-Sit was water soluble and showed high sitravatinib loading and good stability under numerous solution conditions, and was degraded by intracellular esterase to release sitravatinib. In Lewis lung carcinoma (LLC) orthotopic tumor mice, Au@PSMA-Sit enhanced antitumor efficiency by remodeling the TME. Immune profiling with single-cell RNA sequencing showed that Au@PSMA-Sit treatment increased the CD8 T cell cluster and decreased the M2-type macrophage cluster compared to treatment with pure sitravatinib. Au@PSMA-Sit reduced LLC cell proliferation and upgraded M1 polarization of LLC-cocultured TAMs through inhibition of TAM receptors (Tyro3, AXL, and MerTK) after intracellular release of sitravatinib. Au@PSMA-Sit promoted endocytosis-induced endoplasmic reticulum (ER) stress-mediated spleen tyrosine kinase signaling activation, which regulated immunosuppressive TAMs metabolism via enhancement of mitochondrial fission and glycolysis leading to immunogenic modulation. Furthermore, Au@PSMA-Sit enhanced immunogenic cell death through endocytosis/ER stress-mediated release of CRT and HMGB1 from LLC cells, leading to dendritic cell maturation and cytotoxic T cell activation. Therefore, macrophage and CD8 T cell depletion using blocking antibodies diminished the antitumor efficiency of Au@PSMA-Sit. Our results indicate the potential of nano-formulated sitravatinib for strengthening anti-cancer effects in the absence of immunotherapy via immunogenic remodeling of the KRAS-mutant lung TME.

CUL4B functions as a tumor suppressor in KRAS-driven lung tumors by inhibiting the recruitment of myeloid-derived suppressor cells.

Lung cancer is the leading cause of cancer-related death worldwide. KRAS mutations are the most common oncogenic alterations found in lung cancer. Unfortunately, treating KRAS-mutant lung adenocarcinoma (ADC) remains a major oncotherapeutic challenge. Here, we used both autochthonous and transplantable KRAS-mutant tumor models to investigate the role of tumor-derived CUL4B in KRAS-driven lung cancers. We showed that knockout or knockdown of CUL4B promotes lung ADC growth and progression in both models. Mechanistically, CUL4B directly binds to the promoter of Cxcl2 and epigenetically represses its transcription. CUL4B deletion increases the expression of CXCL2, which binds to CXCR2 on myeloid-derived suppressor cells (MDSCs) and promotes their migration to the tumor microenvironment. Targeting of MDSCs significantly delayed the growth of CUL4B knockdown KRAS-mutant tumors. Collectively, our study provides mechanistic insights into the novel tumor suppressor-like functions of CUL4B in regulating KRAS-driven lung tumor development.

UHRF1 is a mediator of KRAS driven oncogenesis in lung adenocarcinoma

KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention.

Senescent alveolar macrophages promote early-stage lung tumorigenesis.

Senescent cells play relevant but context-dependent roles during tumorigenesis. Here, in an oncogenic Kras-driven lung cancer mouse model, we found that senescent cells, specifically alveolar macrophages, accumulate early in neoplasia. These macrophages have upregulated expression of p16INK4a and Cxcr1, are distinct from previously defined subsets and are sensitive to senolytic interventions, and suppress cytotoxic Tcell responses. Their removal attenuates adenoma development and progression in mice, indicating their tumorigenesis-promoting role. Importantly, we found that alveolar macrophages with these properties increase with normal aging in mouse lung and in human lung adenocarcinoma in situ. Collectively, our study indicates that a subset of tissue-resident macrophages can support neoplastic transformation through altering their local microenvironment, suggesting that therapeutic interventions targeting senescent macrophages may attenuate lung cancer progression during early stages of disease.

Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer.

The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.

Abstract A019: The role of KRAS ubiquitination in lung cancer heterogeneity

Abstract Lung cancer is the most frequent cancer with an aggressive clinical course and high mortality rates. About 30% of non-small lung cancer is driven by activating mutations in KRAS (Kirsten rat sarcoma virus). KRAS signaling is tightly controlled through a series of post-transcriptional mechanisms, whereas dysregulation of KRAS activity is translated into heterogeneous clinical behavior. We, and others, have recently implicated leucine zipper-like transcriptional regulator 1 (LZTR1), an adaptor of the CUL3-containing E3 ligase complex, in the control of RAS ubiquitination. Heterozygous loss of LZTR1 is found in one-third of lung adenocarcinomas and co-occurs with oncogenic KRAS mutations, suggesting that LZTR1 loss could contribute to lung cancer by modulating KRAS signaling and affecting the drug response. Both WT and oncogenic KRAS mutant protein levels were modulated by LZTR1 according to a Global protein screen (GPS) scoring regulators of KRAS stability. LZTR1 also directly ubiquitinated mutant KRAS in lung cancer cells. To assess the impact of LZTR1 on KRAS-driven lung cancer, we then used the Kras-G12D mouse model. We found that either homozygous, or heterozygous knockout of Lztr1 cooperates with oncogenic KRAS and promotes tumor initiation and progression at early stages of lung cancer development. The observed phenotype arises from the changes of the immune tumor microenvironment mediated by the differential activation of RAL-B (Ras-related protein Ral-b) signaling in the absence of LZTR1. These results shed a novel light on the crosstalk between dysregulation of KRAS ubiquitination, paracrine communication exerted by the cancer cells through secreted cytokines, and the immune response in lung cancer, which has thwarted the effectiveness of therapy to date. Citation Format: Tonci Ivanisevic. The role of KRAS ubiquitination in lung cancer heterogeneity [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A019.

Long non-coding RNA HIF1A-As2 and MYC form a double-positive feedback loop to promote cell proliferation and metastasis in KRAS-driven non-small cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the main oncogenic driver in lung cancer that can be activated by gene mutation or amplification, but whether long non-coding RNAs (lncRNAs) regulate its activation remains unknown. Through gain and loss of function approaches, we identified that lncRNA HIF1A-As2, a KRAS-induced lncRNA, is required for cell proliferation, epithelial-mesenchymal transition (EMT) and tumor propagation in non-small cell lung cancer (NSCLC) in vitro and in vivo. Integrative analysis of HIF1A-As2 transcriptomic profiling reveals that HIF1A-As2 modulates gene expression in trans, particularly regulating transcriptional factor genes including MYC. Mechanistically, HIF1A-As2 epigenetically activates MYC by recruiting DHX9 on MYC promoter, consequently stimulating the transcription of MYC and its target genes. In addition, KRAS promotes HIF1A-As2 expression via the induction of MYC, suggesting HIF1A-As2 and MYC form a double-regulatory loop to strengthen cell proliferation and tumor metastasis in lung cancer. Inhibition of HIF1A-As2 by LNA GapmeR antisense oligonucleotides (ASO) significantly improves sensitization to 10058-F4 (a MYC-specific inhibitor) and cisplatin treatment in PDX and KRASLSLG12D-driven lung tumors, respectively.

Abstract 5723: An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data

Abstract Recent development of therapies targeting oncogenes have dramatically improved cancer care for subsets of patients and generated a wave of interest in cancer precision medicine. However, effective targeted therapies are scarce as we have a limited understanding of how drug responses are modulated by tumor genotype. Here, we utilize both human data and in vivo models to identify genetic drivers of therapy response in KRAS-driven non-small cell lung cancer patients treated with chemotherapy. We first present an integrative causal inference analysis of three clinical data resources: (1) the recently released Genomics Evidence Neoplasia Information Exchange, Biopharma Collaboration dataset (GENIE-BPC; n=197), (2) a selected set of patients from the Tempus Clinico-genomic Database (n=330), and (3) an additional cohort from Memorial Sloan Kettering Cancer Center (n=218). Each dataset was first analyzed separately using a causal inference pipeline. Doubly robust estimators for each variant were inferred within a counting process survival analysis model accounting for time-varying treatments and immortal time bias. Meta-analysis of the results from all three cohorts identified three commonly mutated and highly replicable genes with a significant effect on overall survival: KEAP1, SMARCA4, and CDKN2A. As further validation, we used our murine in vivo pharmacogenomics (PGx) platform that can quantify the effects of therapies across thousands of tumors of diverse genotypes. These tumors are initiated de novo in the native microenvironmental context in mice with an intact adaptive immune system. We tested a chemotherapy combination of carboplatin and pemetrexed in mice with KrasG12D-driven lung tumors and inactivation of each of 60 putative tumor suppressors. Treatment led to >75% reduction in tumor sizes relative to tumor suppressor inactivated (matched) vehicle-treated controls. These models identified causal effects for two out of the three candidates above: KEAP1 (resistance) and CDKN2A (sensitive). Moreover, our PGx platform identified additional candidate genes beyond those found using the clinical data, which had insufficient sample size for these rarely mutated genes. Together, we demonstrate how leveraging our PGx platform together with human data within a causal inference framework may improve the stratification of patients by their clinical outcomes, profoundly advancing the promise of precision medicine. Citation Format: David Amar, Erick Scott, Ian P. Winters, Gregory D. Wall, Dmitri A. Petrov, Monte M. Winslow, Joseph Juan, Ian K. Lai, Lafia Sebastian, Edwin A. Apilado, Gabriel Grenot, Vy B. Tran, Charles Rudin, Michael J. Rosen. An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5723.

Abstract 486: Characterization of a novel series of highly selective PARP7 inhibitors

Abstract PARP7 is a cellular stress-induced enzyme that adds mono-ADP-ribose groups to a variety of substrate proteins thereby regulating their function. One such substrate is the kinase TBK1 which regulates activity of cGAS-STING and RIG-I nucleic acid sensing pathways. Up-regulation of PARP7 expression in cancers applies a brake to cytosolic nucleic acid sensing and the Type I interferon response. This creates an immunosuppressive tumor microenvironment (TME) leading to faster tumor growth. In addition, inhibition of PARP7 has also been shown to directly arrest growth in a subset of cancer cells via the promotion of a senescence phenotype, inhibition of autophagy and regulation of metabolism. Through the use of structure-based drug design we describe the characterization of potent and selective inhibitors of PARP7. These molecules block viability of a subset of immortalized and primary cancer cells in 2D culture. We have observed potent cGAS-STING independent growth arrest in a number of cancer cell lines harboring a KRAS G12C mutation, and studies combining PARP7 inhibitors with commercially available KRAS G12C inhibitors yield additional in vitro efficacy. Recent studies have shown that inhibitors targeting oncogenic RAS pathway signaling can activate ULK1/2-mediated autophagy as an adaptive treatment resistance mechanism. Hence, our data support further studies investigating the use of PARP7 inhibitors in KRAS-driven cancers either as single agents or in combination with mutant KRAS inhibitors. Our molecules also stimulate robust induction of type I interferon (IFNβ) and downstream chemokines such as CXCL10 in a cGAS-STING pathway dependent manner in a range of cancer cells and 3D organoids. This induction is augmented by combining agonists of cGAS-STING and RIG-I pathways with PARP7 inhibitors. In addition, PARP7 inhibitors work in concert with exogenous DNA-damaging agents such as chemotherapeutics and X-ray radiation to induce the type I interferon response in cancer cells. Overall, these data highlight the opportunity to utilize PARP7 inhibitors in cancers where high genomic instability leads to aberrant cytosolic nucleic acid levels or in concert with exogenous DNA-damaging agents. Our PARP7 inhibitors exhibit highly desirable physico-chemical and in vitro ADME properties coupled with an exceptional in vitro safety profile which translates to best-in-class rodent PK. In a KRAS G12C lung cancer xenograft model (NCI-H1373), twice daily oral administration of an exemplar PARP7 inhibitor drives rapid and full tumor regression in a dose-dependent manner. The anti-tumor effects correlate with robust tumor PK/PD upon measurement of tumor IFNβ and CXCL10. In summary, we describe the characterization of a lead series of highly potent and selective PARP7 inhibitors which demonstrate excellent in vitro ADMET and in vivo PK properties leading to best-in-class anti-tumor efficacy in a KRAS-driven lung cancer xenograft model. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP7 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 486.