Fernando J. de Miguel

Despite the established use of immune checkpoint inhibitors (ICIs) to treat non-small cell lung cancer (NSCLC), only a subset of patients benefit from treatment and ∼50% of patients whose tumors respond eventually develop acquired resistance (AR). To identify novel drivers of AR, we generated murine Msh2 knock-out (KO) lung tumors that initially responded but eventually developed AR to anti-PD-1, alone or in combination with anti-CTLA-4. Resistant tumors harbored decreased infiltrating T cells and reduced cancer cell-intrinsic MHC-I and MHC-II levels, yet remained responsive to IFNγ. Resistant tumors contained extensive regions of hypoxia, and a hypoxia signature derived from single-cell transcriptional profiling of resistant cancer cells was associated with decreased progression-free survival in a cohort of NSCLC patients treated with anti-PD-1/PD-L1 therapy. Targeting hypoxic tumor regions using a hypoxia-activated pro-drug delayed AR to ICIs in murine Msh2 KO tumors. Thus, this work provides a rationale for targeting tumor metabolic features, such as hypoxia, in combination with immune checkpoint inhibition.

Expression of the long noncoding RNA (lncRNA) metastasis–associated lung adenocarcinoma transcript 1 ( MALAT1 ) correlates with tumor progression and metastasis in many tumor types. However, the impact and mechanism of action by which MALAT1 promotes metastatic disease remain elusive. Here, we used CRISPR activation (CRISPRa) to overexpress MALAT1/Malat1 in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. Malat1 overexpression was sufficient to promote the progression of LUAD to metastatic disease in mice. Overexpression of MALAT1/Malat1 enhanced cell mobility and promoted the recruitment of protumorigenic macrophages to the tumor microenvironment through paracrine secretion of CCL2/Ccl2. Ccl2 up-regulation was the result of increased global chromatin accessibility upon Malat1 overexpression. Macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

Upregulated pathways in EGFR mutant cell lines transfected with ASCL1

Changes in chromatin accessibility in HCC827 and PC9 cells upon ASCL1 expression. Global changes in chromatin accessibility in mutant EGFR lung cancer cell lines following ASCL1 overexpression and osimertinib treatment.

Table S2 from ASCL1 Drives Tolerance to Osimertinib in EGFR Mutant Lung Cancer in Permissive Cellular Contexts

Upregulated pathways in EGFR mutant cell lines transfected with ASCL1

Characterization of mutant EGFR lung cancer PDXs at single-cell resolution. Workflow for scRNA-seq on PDXs and single cell data for an additional PDX.

Effects of erlotinib and TKI withdrawal on PDX growth.

ASCL1 expression activates an EMT transcriptional program in permissive cellular contexts. Expression of individual EMT marker genes in mutant EGFR lung cancer cell lines and PDXs with or without ASCL1 overexpression.

Drug tolerant residual disease is characterized by profound gene expression, but not mutational, changes compared to untreated tumors. Pyrosequencing results and mapping of bulk RNA-seq reads to mouse and human genome.