MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex.

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

The MNT transcription factor autoregulates its expression and supports proliferation in MYC-associated factor X (MAX)-deficient cells.

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

BACKGROUND: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. METHODS: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. RESULTS: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. CONCLUSIONS: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

Expression inactivation of SMARCA4 by microRNAs in lung tumors.

SMARCA4 is the catalytic subunit of the SWI/SNF chromatin-remodeling complex, which alters the interactions between DNA and histones and modifies the availability of the DNA for transcription. The latest deep sequencing of tumor genomes has reinforced the important and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer. However, although SWI/SNF complex plays a key role in gene expression, the regulation of this complex itself is poorly understood. Significantly, an understanding of the regulation of SMARCA4 expression has gained in importance due to recent proposals incorporating it in therapeutic strategies that use synthetic lethal interactions between SMARCA4-MAX and SMARCA4-SMARCA2. In this report, we found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3'UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functional relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the aforementioned synthetic lethal therapeutic strategies.

Cell adhesion and polarity in squamous cell carcinoma of the lung.

Lung cancer is a deadly disease that can roughly be classified into three histopathological groups: lung adenocarcinomas, lung squamous cell carcinomas (LSCCs), and small cell carcinomas. These types of lung cancer are molecularly, phenotypically, and regionally diverse neoplasms, reflecting differences in their cells of origin. LSCCs commonly arise in the airway epithelium of a main or lobar bronchus, which is an important line of defence against the external environment. Furthermore, most LSCCs are characterized histopathologically by the presence of keratinization and/or intercellular bridges, consistent with the molecular features of these tumours, characterized by high levels of transcripts encoding keratins and proteins relevant to intercellular junctions and cell polarity. In this review, the relationships between the molecular features of LSCCs and the types of cell and epithelia of origin are discussed. Recurrent alterations in genes involved in intercellular adhesion and cell polarity in LSCCs are also reviewed, emphasizing the importance of the disruption of PAR3 and the PAR complex. Finally, the possible functional effects of these alterations on epithelial homeostasis, and how they contribute to the development of LSCC, are discussed.

A DNA methylation fingerprint of 1628 human samples.

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

HLA-I levels correlate with survival outcomes in response to immune checkpoint inhibitors in non-small cell lung cancer.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) have provided a breakthrough in the treatment of non-small cell lung cancer (NSCLC) patients, but only some patients benefit substantively. Identifying definitive predictive biomarkers could overcome this limitation. MATERIALS AND METHODS: We selected 146 metastatic NSCLC patients treated with anti-PD-(L)1. Immunohistochemistry of HLA-I, PD-L1 and CD73 was performed in 122 tumor biopsies at diagnosis. The association with patients, tumor parameters, and the predictive value to ICI treatment were determined. RESULTS: In our cohort, 42 %, 25 %, and 21 % of the tumors exhibited high levels of HLA-I, PD-L1, and CD73, respectively. Lung adenocarcinomas displayed elevated CD73 levels, compared with lung squamous cell carcinomas (P = 0.026). High PD-L1 was significantly correlated with high levels of HLA-I (P = 0.005) and of CD73 (P = 0.025). Patients with high-level HLA-I tumors exhibited more favorable clinical outcomes following ICI, with a median overall survival of 30.7 months (95 % confidence interval [CI]: 18.3 months-not reached), compared with 18.2 months (95 % CI: 12.4-25.2 months) in patients with low-level HLA-I tumors (P = 0.016). The median progression-free survival (PFS) for patients with high-level HLA-I tumors was 18.5 months (95 % CI: 11.1-57.1 months), longer than patients with low-level HLA-I tumors, whose median PFS was 9.2 months (95 % CI: 7.2-11.9 months) (P = 0.006). In a multivariable analysis, high-level HLA-I was independently associated with lower risk of progression to ICI (HR = 0.46, 95 % CI 0.24-0.87; P = 0.018). CONCLUSIONS: High-level HLA-I were associated with better clinical outcomes to ICI in our cohort of NSCLC patients. Therefore, further investigations are warranted to refine this biomarker and validate its efficacy in prospective and larger set of patients.

Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.

Growth factor receptors (GFRs) are amenable to therapeutic intervention in cancer and it is important to select patients appropriately. One of the mechanisms for activation of GFRs is gene amplification (GA) but discrepancies arising from the difficulties associated with data interpretation and the lack of agreed parameters confound the comparison of results from different laboratories. Here, we attempt to establish appropriate conditions for standardization of the determination of GA in a panel of GFRs. A NSCLC tissue microarray panel containing 302 samples was screened for alterations at ALK, FGFR1, FGFR2, FGFR3, ERBB2, IGF1R, KIT, MET and PDGFRA by FISH, immunostaining and/or real-time quantitative RT-PCR. Strong amplification was found for FGFR1, ERBB2, KIT/PDFGRA and MET, with frequencies ranging from 1 to 6%. Thresholds for overexpression and GA were established. Strong immunostaining was found in most tumors with ERBB2, MET and KIT amplification, although some tumors underwent strong immunostaining in the absence of GA. KIT and PDFGRA were always coamplified, but only one tumor showed PDGFRA overexpression, indicating that KIT is the main target. Amplification of FGFR1 predominated in squamous cell carcinomas, although the association with overexpression was inconclusive. Interestingly, alterations at ALK, MET, EGFR, ERBB2 and KRAS correlated with augmented levels of phospho-S6 protein, suggesting activation of the mTOR pathway, which may prove useful to pre-select tumors for testing. Overall, here, we provide with parameters for the determination of GA at ERBB2, MET, KIT and PDGFRA which could be implemented in the clinic to stratify lung cancer patients for specific treatments.

Epigenetic disruption of cadherin-11 in human cancer metastasis.

Little is known about the molecular events occurring in the metastases of human tumours. Epigenetic alterations are dynamic lesions that change over the natural course of the disease, and so they might play a role in the biology of cancer cells that have departed from the primary tumour. Herein, we have adopted an epigenomic approach to identify some of these changes. Using a DNA methylation microarray platform to compare paired primary tumour and lymph node metastatic cell lines from the same patient, we observed cadherin-11 promoter CpG island hypermethylation as a likely target of the process. We found that CDH11 DNA methylation-associated transcriptional silencing occurred in the corresponding lymph node metastases of melanoma and head and neck cancer cells but not in the primary tumours. Using in vitro and in vivo cellular and mouse models for depleted or enhanced CDH11 activity, we also demonstrated that CDH11 acts as an inhibitor of tumour growth, motility and dissemination. Most importantly, the study of CDH11 5'-CpG island hypermethylation in primary tumours and lymph node metastases of cancer patients showed this epigenetic alteration to be significantly confined to the disseminated cells. Overall, these results indicate the existence of metastasis-specific epigenetic events that might contribute to the progression of the disease.

Characterization of a cohort of metastatic lung cancer patients harboring KRAS mutations treated with immunotherapy: differences according to KRAS G12C vs. non-G12C.

Approximately 20% of lung adenocarcinomas harbor activating mutations at KRAS, an oncogene with the ability to alter the tumor immune microenvironment. In this retrospective study, we examined 103 patients with KRAS-mutant lung adenocarcinoma who were treated with immunotherapy-based regimens and we evaluated the clinical outcomes according to PD-L1 expression and the type of KRAS mutation. Among all patients included, 47% carried KRAS G12C mutation whereas 53% harbored KRAS non-G12C mutations. PD-L1 status was available for 77% of cases, with higher expression among KRAS G12C tumors (p = 0.01). Better overall survival and progression-free survival were observed in high PD-L1 expression tumors, regardless of KRAS mutation type. The heterogeneous nature of KRAS-mutant tumors and the presence of other co-mutations may contribute to different outcomes to immunotherapy-based strategies.

MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer.

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC.

Biological and clinical perspectives of the actionable gene fusions and amplifications involving tyrosine kinase receptors in lung cancer.

Identifying molecular oncogenic drivers is crucial for precision oncology. Genetic rearrangements, including gene fusions and gene amplification, involving and activating receptor tyrosine kinases (RTKs) are recurrent in solid tumors, particularly in non-small cell lung cancer. Advances in the tools to detect these alterations have deepened our understanding of the underlying biology and tumor characteristics and have prompted the development of novel inhibitors targeting activated RTKs. Nowadays, druggable oncogenic rearrangements are found in around 15% of lung adenocarcinomas. However, taken separately, each of these alterations has a low prevalence, which poses a challenge to their diagnosis. The identification and characterization of novel targetable oncogenic rearrangements in lung cancer continue to expand, as shown by the recent discovery of the CLIP1-LTK fusion found in 0.4% of lung adenocarcinomas. While tyrosine kinase inhibitors that block the activity of RTKs have represented a breakthrough in the therapeutic landscape by improving the prognosis of this disease, prolonged treatment inevitably leads to the development of acquired resistance. Here, we review the oncogenic fusions and gene amplifications involving RTK in lung cancer. We address the genetic and molecular structure of oncogenic RTKs and the methods to diagnose them, emphasizing the role of next-generation sequencing technologies. Furthermore, we discuss the therapeutic implications of the different tyrosine kinase inhibitors, including the current clinical trials and the mechanisms responsible for acquired resistance. Finally, we provide an overview of the use of liquid biopsies to monitor the course of the disease.

Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors.

The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra-deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra-deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic-fluorescence-based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.

The SRY-HMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer.

The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared DNA copy number and mRNA expression levels in lung cancer cell lines. We identified several amplicons (5p13, 6p22-21, 11q13, 17q21 and 19q13) that had a concomitant increase in gene expression. These regions were also found to be amplified in lung primary tumours. We mapped the boundaries and measured expression levels of genes within the chromosome 6p amplicon. The Sry-HMG box gene SOX4 (sex-determining region Y box 4), which encodes a transcription factor involved in embryonic cell differentiation, was overexpressed by a factor of 10 in cells with amplification relative to normal cells. SOX4 expression was also stronger in a fraction of lung primary tumours and lung cancer cell lines and was associated with the presence of gene amplification. We also found variants of SOX4 in lung primary tumours and cancer cell lines, including a somatic mutation that introduced a premature stop codon (S395X) at the serine-rich C-terminal domain. Although none of the variants increased the transactivation ability of SOX4, overexpression of the wildtype and of the non-truncated variants in NIH3T3 cells significantly increased the transforming ability of the weakly oncogenic RHOA-Q63L. In conclusion, our results show that, in lung cancer, SOX4 is overexpressed due to gene amplification and provide evidence of oncogenic properties of SOX4.

Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer.

The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

A catalog of genes homozygously deleted in human lung cancer and the candidacy of PTPRD as a tumor suppressor gene.

A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis.

Targeting KRAS in Lung Cancer Beyond KRAS G12C Inhibitors: The Immune Regulatory Role of KRAS and Novel Therapeutic Strategies.

Approximately 20% of lung adenocarcinomas harbor KRAS mutations, an oncogene that drives tumorigenesis and has the ability to alter the immune system and the tumor immune microenvironment. While KRAS was considered "undruggable" for decades, specific KRAS G12C covalent inhibitors have recently emerged, although their promising results are limited to a subset of patients. Several other drugs targeting KRAS activation and downstream signaling pathways are currently under investigation in early-phase clinical trials. In addition, KRAS mutations can co-exist with other mutations in significant genes in cancer (e.g., STK11 and KEAP1) which induces tumor heterogeneity and promotes different responses to therapies. This review describes the molecular characterization of KRAS mutant lung cancers from a biologic perspective to its clinical implications. We aim to summarize the tumor heterogeneity of KRAS mutant lung cancers and its immune-regulatory role, to report the efficacy achieved with current immunotherapies, and to overview the therapeutic approaches targeting KRAS mutations besides KRAS G12C inhibitors.

Molecular profiling of long-term responders to immune checkpoint inhibitors in advanced non-small cell lung cancer.

Immunotherapy has transformed advanced non-small cell lung cancer (NSCLC) treatment strategies and has led to unprecedented long-lasting responses in some patients. However, the molecular determinants driving these long-term responses remain elusive. To address this issue, we performed an integrative analysis of genomic and transcriptomic features of long-term immune checkpoint inhibitors (ICIs)-associated responders. We assembled a cohort of 47 patients with NSCLC receiving ICIs that was enriched in long-term responders [>18 months of progression-free survival (PFS)]. We performed whole-exome sequencing from tumor samples, estimated the tumor mutational burden (TMB), and inferred the somatic copy number alterations (SCNAs). We also obtained gene transcription data for a subset of patients using Nanostring, which we used to assess the tumor immune infiltration status and PD-L1 expression. Our results indicate that there is an association between TMB and benefit to ICIs, which is driven by those patients with long-term response. Additionally, high SCNAs burden is associated with poor response and negatively correlates with the presence of several immune cell types (B cells, natural killers, regulatory T cells or effector CD8 T cells). Also, CD274 (PD-L1) expression is increased in patients with benefit, mainly in those with long-term response. In our cohort, combined assessment of TMB and SCNAs burden enabled identification of long-term responders (considering PFS and overall survival). Notably, the association between TMB, SCNAs burden, and PD-L1 expression with the outcomes of ICIs treatment was validated in two public datasets of ICI-treated patients with NSCLC. Thus, our data indicate that TMB is associated with long-term benefit following ICIs treatment in NSCLC and that TMB, SCNAs burden, and PD-L1 are complementary determinants of response to ICIs.

SMARCA4 deficient tumours are vulnerable to KDM6A/UTX and KDM6B/JMJD3 blockade.

Despite the genetic inactivation of SMARCA4, a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. SMARCA4-mutant cells also show an impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, and a strong dependency on these histone demethylases, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4-mutant lung cancer cells or primary small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT), had strong anti-tumour effects. In this work we highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4-mutant cancer patients.