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.

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.

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.

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. Keywords: MAX dimerization protein MLX; MAX network transcriptional repressor (MNT); MXD family; MYC-associated factor X (MAX); Myc (c-Myc); basic helix-loop-helix leucine zipper protein; gene regulation; proliferation; promoter; transcription.

Genomic analysis of lung adenocarcinomas has revealed that the MGA gene, which encodes a heterodimeric partner of the MYC-interacting protein MAX, is significantly mutated or deleted in lung adenocarcinomas. Most of the mutations are loss of function for MGA, suggesting that MGA may act as a tumor suppressor. Here, we characterize both the molecular and cellular role of MGA in lung adenocarcinomas and illustrate its functional relevance in the MYC pathway. Although MGA and MYC interact with the same binding partner, MAX, and recognize the same E-box DNA motif, we show that the molecular function of MGA appears to be antagonistic to that of MYC. Using mass spectrometry-based affinity proteomics, we demonstrate that MGA interacts with a noncanonical PCGF6-PRC1 complex containing MAX and E2F6 that is involved in gene repression, while MYC is not part of this MGA complex, in agreement with previous studies describing the interactomes of E2F6 and PCGF6. Chromatin immunoprecipitation-sequencing and RNA sequencing assays show that MGA binds to and represses genes that are bound and activated by MYC. In addition, we show that, as opposed to the MYC oncoprotein, MGA acts as a negative regulator for cancer cell proliferation. Our study defines a novel MYC/MAX/MGA pathway, in which MYC and MGA play opposite roles in protein interaction, transcriptional regulation, and cellular proliferation. IMPLICATIONS: This study expands the range of key cancer-associated genes whose dysregulation is functionally equivalent to MYC activation and places MYC within a linear pathway analogous to cell-cycle or receptor tyrosine kinase/RAS/RAF pathways in lung adenocarcinomas.

Background: The etiology and the molecular basis of lung adenocarcinomas (LuADs) in nonsmokers are currently unknown. Furthermore, the scarcity of available primary cultures continues to hamper our biological understanding of non-smoking-related lung adenocarcinomas (NSK-LuADs). Patients and methods: We established patient-derived cancer cell (PDC) cultures from metastatic NSK-LuADs, including two pairs of matched EGFR-mutant PDCs before and after resistance to tyrosine kinase inhibitors (TKIs), and then performed whole-exome and RNA sequencing to delineate their genomic architecture. For validation, we analyzed independent cohorts of primary LuADs. Results: In addition to known non-smoker-associated alterations (e.g. RET, ALK, EGFR, and ERBB2), we discovered novel fusions and recurrently mutated genes, including ATF7IP, a regulator of gene expression, that was inactivated in 5% of primary LuAD cases. We also found germline mutations at dominant familiar-cancer genes, highlighting the importance of genetic predisposition in the origin of a subset of NSK-LuADs. Furthermore, there was an over-representation of inactivating alterations at RB1, mostly through complex intragenic rearrangements, in treatment-naive EGFR-mutant LuADs. Three EGFR-mutant and one EGFR-wild-type tumors acquired resistance to EGFR-TKIs and chemotherapy, respectively, and histology on re-biopsies revealed the development of small-cell lung cancer/squamous cell carcinoma (SCLC/LuSCC) transformation. These features were consistent with RB1 inactivation and acquired EGFR-T790M mutation or FGFR3-TACC3 fusion in EGFR-mutant tumors. Conclusions: We found recurrent alterations in LuADs that deserve further exploration. Our work also demonstrates that a subset of NSK-LuADs arises within cancer-predisposition syndromes. The preferential occurrence of RB1 inactivation, via complex rearrangements, found in EGFR-mutant tumors appears to favor SCLC/LuSCC transformation under growth-inhibition pressures. Thus RB1 inactivation may predict the risk of LuAD transformation to a more aggressive type of lung cancer, and may need to be considered as a part of the clinical management of NSK-LuADs patients. Keywords: EGFR; RB1; lung adenocarcinoma; nonsmokers; tyrosine kinase inhibitors; whole-exome sequencing.

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.

The incorporation into clinical practice of immune-checkpoint inhibitors (ICIs), such as those targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death 1 (PD-1) and its ligand (PD-L1), has represented a major breakthrough in non-small cell lung cancer (NSCLC) treatment, especially in cases where the cancer has no druggable genetic alterations. Despite becoming the standard of care in certain clinical settings, either alone or in combination with chemotherapy, a proportion of patients do not respond while others actually progress during treatment. Therefore, there is a clinical need to identify accurate predictive biomarkers and to develop novel therapeutic strategies based on ICIs. Although they have limitations, the current markers evaluated to select which patients will undergo ICI treatment are the levels of PD-L1 and the tumor mutational burden. In this paper we describe what is currently known about the dynamic interaction between the cancer cell and the immune system during carcinogenesis, with a particular focus on the description of the functions and gene alterations that preclude the host immunoresponse in NSCLC. We emphasize the deleterious gene alterations in components of the major histocompatibility complex (HLA-I or B2M) and of the response to IFNγ (such as JAK2) which are mutually exclusive and can affect up to one fifth of the NSCLCs. The participation of other gene alterations, such as those of common oncogenes and tumor suppressors, and of the epigenetic alterations will also be discussed, in detail. Finally, we discuss the potential use of the tumor's genetic profile to predict sensitivity to ICIs.