Abstract Loss of function (LOF) of the serine-threonine kinase STK11/LKB1, which has been reported in ~15% of non-small cell lung cancer (NSCLC), drives resistance to immune checkpoint therapies such as PD1. In vivo CRISPR screens in tumor-bearing immune competent mice identified the Corepressor of Repressor Element 1 Silencing Transcription (CoREST) complex as a synthetic lethal immune evasion target for STK11- mutant tumors. Here, we demonstrate that TNG260, a small molecule drug specifically targeting the CoREST complex, can sensitize STK11 mutant lung cancers to anti-PD1 immunotherapy. Combination treatment with TNG260 and anti-PD1 arrested KRAS/STK11 mutant NSCLC tumor growth with no major toxicity observed, as confirmed in both allograft and genetically engineered mouse models (GEMMs) of STK11 mutant NSCLC. Further transcriptional analysis via bulk RNA-seq revealed that treatment of KRAS/STK11 mutant lung tumors with TNG260 promotes genes related to inflammatory responses and suppresses genes related to cell cycle and mitotic checkpoint(s). This phenotype was not observed in STK11 wildtype KRAS/P53 mutant lung tumors. Furthermore, combinational treatment of TNG260 with PD1 antibody showed increased macrophages, neutrophils and dendritic cells signatures in vivo. Our data suggests that epigenetic reprogramming via TNG260 sensitizes STK11 mutant NSCLC tumors to anti- PD1 treatment via targeting of the CoREST complex. This work identifies new vulnerabilities in STK11 LOF cancers that can be exploited therapeutically. TNG260 is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492), including NSCLC. Citation Format: Ayushi Patel, Soumyadip Sahu, Ke Geng, Salman Punekar, Janaye Stephens, Jiehui Deng, Ting Chen, Leanne Ahronian, Minjie Zhang, Jannik Andersen, Brian Haines, Kwok-Kin Wong. TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy [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 3916.

Supplementary Figure Legends

Supplementary Table S10 Differential gene expression analysis of scRNA-seq data from tumor cluster in patient #1778 (AMG-510-resistant tumor) vs patient #1566 (control, KRASG12V tumor)

Supplementary Table S11 Differential gene expression analysis of scRNA-seq data from tumor cluster in patient #2349 (MRTX-849/TNO155-resistant) vs patient #1566 (control, KRASG12V tumor)

Supplementary Table S8 RNA-seq analysis of MTRX-849/SHP099-resistant tumor nodules from KCL mice

Supplementary Table S7 RPPA from MTRX-849-resistant tumor nodules from KCL mice

Supplementary Table S3 RNA-seq analysis of H2030 and H2122 cells treated with MRTX-849 or vehicle for 48 hr

Abstract Non–small lung cancers (NSCLC) frequently (∼30%) harbor KRAS driver mutations, half of which are KRASG12C. KRAS-mutant NSCLC with comutated STK11 and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapy. Development of KRASG12C inhibitors (G12Ci) provided a major therapeutic advance, but resistance still limits their efficacy. To identify genes whose deletion augments efficacy of the G12Cis adagrasib (MRTX-849) or adagrasib plus TNO155 (SHP2i), we performed genome-wide CRISPR/Cas9 screens on KRAS/STK11-mutant NSCLC lines. Recurrent, potentially targetable, synthetic lethal (SL) genes were identified, including serine–threonine kinases, tRNA-modifying and proteoglycan synthesis enzymes, and YAP/TAZ/TEAD pathway components. Several SL genes were confirmed by siRNA/shRNA experiments, and the YAP/TAZ/TEAD pathway was extensively validated in vitro and in mice. Mechanistic studies showed that G12Ci treatment induced gene expression of RHO paralogs and activators, increased RHOA activation, and evoked ROCK-dependent nuclear translocation of YAP. Mice and patients with acquired G12Ci- or G12Ci/SHP2i-resistant tumors showed strong overlap with SL pathways, arguing for the relevance of the screen results. These findings provide a landscape of potential targets for future combination strategies, some of which can be tested rapidly in the clinic. Significance: Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005

Supplementary Table S1 MaGeCK analysis of MRTX-849 CRISPR/Cas9 SL screens of NSCLC cell lines

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP7 (5-diphospho-inositol-1,2,3,4,6-pentakisphosphate). Quantitative confocal imaging of the outer nucleolar granular region revealed its expansion when cellular 5-InsP7 levels were elevated by either (a) reducing the 5-InsP7 metabolism by a CRISPR-based knockout (KO) of either NUDT3 or PPIP5Ks; or (b), the heterologous expression of wild-type inositol hexakisphosphate kinase, i.e., IP6K2; separate expression of a kinase-dead IP6K2 mutant did not affect granular volume. Conversely, the nucleolar granular region in PPIP5K KO cells shrank back to the wild-type volume upon attenuating 5-InsP7 synthesis using either a pan-IP6K inhibitor or the siRNA-induced knockdown of IP6K1+IP6K2. Significantly, the inner fibrillar volume of the nucleolus was unaffected by 5-InsP7. We posit that 5-InsP7 acts as an ‘electrostatic glue’ that binds together positively charged surfaces on separate proteins, overcoming mutual protein–protein electrostatic repulsion the latter phenomenon is a known requirement for the assembly of a non-membranous biomolecular condensate.

Supplementary Table S2 Overlapping dropouts (FDR <0.1) from MRTX-849 CRISPR/Cas9 screens in NSCLC cell lines

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)—key downstream effectors of LKB1— induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE Loss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

ABSTRACT LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD + degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD + . Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)—key downstream effectors of LKB1— induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE Loss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

Supplementary Table S9 RPPA data from MRTX-849/SHP099-resistant tumor nodules in KCL mice

Supplementary Table S6 RNA-seq analysis of MTRX-849-resistant tumor nodules from KCL mice

Supplementary Figure from DNA Methylation Profiling Identifies Subgroups of Lung Adenocarcinoma with Distinct Immune Cell Composition, DNA Methylation Age, and Clinical Outcome

Supplementary Figures 1-7 including Figure legends

Introduction: In KRAS-mutant NSCLC, co-occurring alter-ations in LKB1 confer a negative prognosis compared with other mutations such as TP53. LKB1 is a tumor suppressor that coordinates several signaling pathways in response to energetic stress. Our recent work on pharmacologic and genetic inhibition of histone deacetylase 6 (HDAC6) revealed the impaired activity of numerous enzymes involved in glycolysis. On the basis of these previous findings, we explored the therapeutic window for HDAC6 inhibition in metabolically-active KRAS-mutant lung tumors.Methods: Using cell lines derived from mouse autochtho-nous tumors bearing the KRAS/LKB1 (KL) and KRAS/TP53 mutant genotypes to control for confounding germline and somatic mutations in human models, we characterize the metabolic phenotypes at baseline and in response to HDAC6 inhibition. The impact of HDAC6 inhibition was measured on cancer cell growth in vitro and on tumor growth in vivo.Results: Surprisingly, KL-mutant cells revealed reduced levels of redox-sensitive cofactors at baseline. This is asso-ciated with increased sensitivity to pharmacologic HDAC6 inhibition with ACY-1215 and blunted ability to increase compensatory metabolism and buffer oxidative stress. Seeking synergistic metabolic combination treatments, we found enhanced cell killing and antitumor efficacy with glutaminase inhibition in KL lung cancer models in vitro and in vivo.Conclusions: Exploring the differential metabolism of KL and KRAS/TP53-mutant NSCLC, we identified decreased metabolic reserve in KL-mutant tumors. HDAC6 inhibition exploited a therapeutic window in KL NSCLC on the basis of a diminished ability to compensate for impaired glycolysis, nominating a novel strategy for the treatment of KRAS- mutant NSCLC with co-occurring LKB1 mutations.& COPY; 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Objective: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE −/− and TSP-1 −/− /ApoE −/− double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE −/− mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE −/− mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE −/− ; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE −/− on western diet, independent of plasma lipid alterations. Conclusions: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Inositol pyrophosphates (PP-InsPs) comprise an evolutionarily ancient family that exhibit many regulatory roles throughout the eukaryotic kingdoms. These molecules have the extraordinary feature of possessing the most concentrated three-dimensional array of phosphate groups in Nature. This characteristic endows the PP-InsPs with a palette of physicochemical properties, each of which can be separately called upon to serve a wide range of signaling situations. In this review we will illustrate how this unusual, mechanistic flexibility has a consistent functional theme that PP-InsPs regulate multiple aspects of bioenergetic and metabolic homeostasis, in animals, plants and fungi. We will also describe the small-molecule kinases and phosphatases that are responsible for switching on and off PP-InsP signals; these enzymes have a number of rare properties that can finely tune PP-InsP turnover in response to changing environmental conditions. Finally, we will draw attention to important uncertainties in this wide-ranging field of PP-InsP research that remain to be resolved.