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Purpose: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Prostaglandin E2 (PGE 2 ) and 16,16-dimethyl-PGE 2 (dmPGE 2 ) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122 , 3074–3081(2013); W. Goessling et al. Cell Stem Cell 8 , 445–458 (2011); W. Goessling et al. Cell 136 , 1136–1147 (2009)]. Here, we report that PGE 2 -induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE 2 -inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE 2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE 2 , lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist–induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Supplementary Data from Activation of Tumor-Cell STING Primes NK-Cell Therapy

CRISPR screen in tumor-infiltrating T cells

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity.Significance:SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack.Significance:Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell–intrinsic type 1 interferon response.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Table S1

Supplementary Figures 1-12

Contains 16 supplementary figures and references

Supplementary Figure 3: PPP1CB-ALK fusion is oncogenic and sensitive to targeted therapy. A. Immunoblot analysis of ALK downstream signaling proteins in cortical mNSC (CTX#6) and brainstem mNSC (BS#3) expressing PPP1CB-ALK. Cells were treated for 4 hours with ceritinib and lorlatinib at the indicated concentrations.B. Left panel: Soft agar colony-forming assay using stable mNSC PPP1CB-ALK demonstrated colony formation with dose-dependent inhibition by ceritinib. mNSC KRAS did not respond to ceritinib validating the drug specificity. Right panel: Quantification of colony formation under targeted drug inhibition using CellProfiler. Values represent colony counts relative to the untreated group ± s.d. The mean of three independent replicates is shown. Significance between treatments determined by the Mann-Whitney test. *P < 0.05, **P < 0.01.C. Cell viability of ceritinib-treated PPP1CB-ALK NIH-3T3 relative to eGFP-positive cells by CellTiterGlo.D. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 ceritinib-treated cells versus DMSO control.F. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.G. Pharmacodynamic analysis of NIH 3T3-PPP1CB-ALK s.c. tumors treated with vehicle or ceritinib at 30mg/kg for 5 days. Representative H&E stain, ALK, pSTAT3, pAKT S473 and Ki-67 IHC.H. Quantification of Ki-67-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.I. Quantification of pSTAT3-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.J. Tumor growth following subcutaneous implantation of PPP1CB-ALK and eGFP-expressing fibroblasts.K. Kaplan-Meier survival curves of SCID mice injected subcutaneously with mNSC CTX-PPP1CB-ALK (red, n = 5) or mNSC CTX-eGFP (black, n = 4) and treated with ceritinib at 30mg/kg/d for 15 days. Mice were euthanized at endpoint (tumor volume of 2000mm3). The grey area represents the treatment period.L. Vehicle or ceritinib-treated mouse weights at start and end of treatment.

Supplementary Figure 5: Single-cell RNA Sequencing and IHC examination of ALK in human neural development and immature neural cells. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (cortex-dev.cells.ucsc.edu) shows that ALK transcript appears to be enriched in a certain cell type. Color-coded by ALK gene expression (beige to red) and age in weeks (rainbow).B. ALK-stained sagittal section of normal human cerebellum at 15 postconceptional weeks (pcw).C. ALK-stained sagittal section of human fetal neural stem cell pellets at 13 postconceptional weeks (pcw).D. ALK-stained sagittal section of human fetal neural stem cell pellets at 15 postconceptional weeks (pcw).

Purpose:Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established.Experimental Design:We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations.Results:ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib.Conclusions:These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Supplementary Figure 6: Single cell RNA sequencing comparison of ALK expression and other infant/pediatric glioma kinase drivers in developing human and mouse brain. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (http://cells.ucsc.edu/?ds=cortex-dev) shows that ALK positive cells make up less than 1% of all cells in the dataset and aren't localized within a specific cluster.B. Cluster-level summary of the human scRNA dataset shows that ALK expression (TPM) is decreased compared to EGFR, NTRK2, and NTRK3, and ALK positive cells make up a much lower percentage of each cluster compared to those 3 genes.C. 2D representation of developmental mouse forebrain (E12.5 – P6) in a single cell RNA-seq atlas (http://cc-shiny-01.functionalgenomics.ca/braindex/clusters) shows similar results as seen in the human scRNA Nowakowski dataset.D. Cluster-level summary of the mouse scRNA dataset corroborates results seen in the Nowkowski scRNA dataset; that Alk expression (log-normalized) is decreased, and less prevalent than EGFR, NTRK2, and NTRK3.E. 2D representation of RNA sequencing data for NTRK1-3 genes in the Nowakowski developing human cortex dataset. Compared to ALK expression, the NTRK genes have increased expression levels, increased prevalence in the dataset, and show increased localization to specific cluster.F. Similar to (E), the 2D representation of Ntrk genes in the developing mouse scRNA dataset shows increased expression and prevalence compared to Alk.

Supplementary Figure 2: T1 post-contrast axial and sagittal images of ALK-fused and non ALK-altered GBMs and ALK staining in ALK-fused congenital GBMs. A. Example images from patients with ALK aberrations (76, 89, 116, 197)B. In contrast to those without ALK aberrations (right columns). Contrast is gadolinium for those where applicable and show enhancement within both the tumor categories.C. Representative H&E and ALK IHCs of congenital GBM harboring an ALK fusion.

Supplementary Figure 4: Characterization of the lung cancer recurrent DCTN1-ALK fusion identified in cGBM (case ALK.232). A. Integrative Genomic Viewer (IGV) screenshot of the DCTN1-ALK variant.B. Predicted sequence of DCTN1-ALK fusion (DCTN1 exon 1-26, ALK exon 20-29 conserved).C. PDX DCTN1-ALK sub-renal capsule engraftment confimed by IHC.C. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 ceritinib-treated cells versus DMSO control.D. Relative viability of BT164 and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.F. PDX DCTN1-ALK sub-renal capsule engraftment confirmed by IHC.

Supplementary Figure 1: Characterization of the novel LRRFIP1-ALK fusion (case ALK.223) A. Visualization of the read coverage at the fusion sites in ALK and LRRFIP1 genes using Integrative Genomic Viewer.B. Pile-up plot generated by SvABA using whole-genome sequencing data shows the breakpoint coordinates.C. Copy number ratio profile by WGS showing no somatic copy number alterations anywhere in the genome. The x-axis represents the chromosomes.D. Copy number ratio profile on chromosome 2 showing no copy number change of ALK and LRRFIP1 genes.E. Two RT-PCR products of 186bp (variant 1) and 189bp (variant 3) were detected by agarose gel electrophoresis with total RNA from tumor specimen ALK.223.F. Primer sequences of ALK-LRRFIP1 (variants 1 and 2) and LRRFIP1-ALK (variant 3) and the predicted DNA product size.G. Western Blot analysis of ALK signaling pathway in subcutaneous tumors treated with vehicle or lorlatinib. Normal brain was used as control.H. Representative 40x images and CellProfiler quantification of Cleaved Caspase 3- and Ki-67- stained subcutaneous tumors.

Additional information tables 1 to 8.

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. Significance: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

LISA analysis of master regulators of differentially expressed genes

Supplementary Data including supplementary methods, supplementary figures, and supplementary figure legends.

GSEA Hallmarks analysis of RNA-seq of bulk lung tumors

RNA-seq data of bulk lung tumors from sgControl RPP, sgNotch1 RPP, sgNotch2 RPP, and sgAscl1 RPP CRISPR-based SCLC GEMMs

RNA-seq data of the SCLC adherent cells that are either Notch-WT or Notch-Mutant

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations.Significance:A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies1,2. The mechanism of gene regulation in response to dmPGE2 is poorly understood. Here, we show that dmPGE2 regulates inflammatory gene induction by modulating the chromatin architecture and activity of enhancer elements in human HSPCs. We identified the specific genomic reorganization at stimuli-responsive enhancers that permits rapid transcriptional activation. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes that are permissive to binding of the transcription factor CREB. CREB binding to enhancer nucleosomes is concomitant with deposition of the histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone variant nucleosomes retained within inducible enhancers facilitate transcription factor (TF) binding. Acetylation of histone variant nucleosomes by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism by which inflammatory mediators such as dmPGE2 lead to acute transcriptional changes and alter HSPC behavior.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist–induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that accounts for ~15% of lung cancers. While nearly all SCLCs are genetically driven by near universal loss of function (LOF) mutations in RB1 and TP53; several recent studies show that there are different phenotypic SCLC molecular subtypes characterized by expression of lineage transcription factors. These include the neuroendocrine ASCL1 and NEUROD1 subtypes which together comprise ~70-80% of SCLCs. Initially subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoral subtype heterogeneity and plasticity between subtypes. A recent study found that 35-40% of human SCLCs express both ASCL1 and NEUROD1, but the mechanisms driving ASCL1 and NEUROD1 intra-tumoral heterogeneity are not well understood. My laboratory previously developed an autochthonous CRISPR-based SCLC genetically-engineered mouse model (GEMM) generated by intratracheally injecting adenoviruses encoding Cre recombinase and sgRNAs targeting Rb1, Trp53, and Rbl2. Cre turns on Cas9 expression and allows for CRISPR/Cas9 editing of Rb1, Trp53, and Rbl2 in somatic cells in the lungs. The unique advantage of this model is that it allows the inclusion of sgRNAs targeting additional genes of interest in the same adenovirus. Using this CRISPR-based autochthonous SCLC GEMM approach, we studied the consequences of inactivating the epigenetic modifier KDM6A during SCLC tumorigenesis. KDM6A functions as an H3K27 histone demethylase and also exists in the COMPASS complex with KMT2C/D to promote H3K4 mono-/di-methylation at enhancers. KDM6A along with its protein binding partner KMT2D are mutated in SCLC and KDM6A has been implicated in controlling differentiation in other lineages. Strikingly, we found that KDM6A inactivation in SCLC GEMMs induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumors that expressed both ASCL1 and NEUROD1. ATAC-sequencing showed open chromatin at the promoters of NEUROD1 and NEUROD1 target genes in KDM6A inactivated tumors. Interestingly, KDM6A inactivated tumors showed a spectrum of ASCL1 to NEUROD1 heterogeneity where some KDM6A inactivated tumors completely lost ASCL1 and solely expressed NEUROD1, some tumors expressed ASCL1 and NEUROD1 in a mutually exclusive manner, while others primarily expressed ASCL1 with very few NEUROD1 positive cells. Mechanistically, KDM6A binds and maintains ASCL1 target genes in an active chromatin state with its loss increasing H3K27me3 near both promoters and enhancers, and decreasing H3K4me1/2 at enhancers together leading to a cell state primed for ASCL1 to NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC GEMM to model ASCL1 and NEUROD1 subtype heterogeneity, which is found in 35-40% of human SCLCs. Citation Format: Leslie Duplaquet, Yixiang Li, Matthew A. Booker, Yingtian Xie, Radhika A. Patel, Deli Hong, Thomas Denize, Emily Walton, Yasmin N. Laimon, Roderick Bronson, Jackson Southard, Shuqiang Li, Sabina Signoretti, Michael Y. Tolstorukov, Paloma Cejas, Henry W. Long, Michael C. Haffner, Matthew G. Oser. Small cell lung cancer subtype plasticity is regulated by KDM6A. [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 5774.

Loss of Function (LOF) mutations in the NOTCH receptors are found in several different cancers most notably in small cell lung cancer (SCLC) and squamous cell carcinomas (SCC). We previously developed genetically engineered mouse models (GEMM) of SCLC using CRISPR/Cas9 engineered to be NOTCH1-Mutant, NOTCH2-Mutant, and NOTCH-WT. Synthetic lethality provides a paradigm for targeting cancers with LOF mutations in tumor suppressor genes. In applying this paradigm, one looks for specific vulnerabilities that are created upon loss of the gene of interest. Using 6 cell lines developed from these CRISPR-based SCLC GEMMs which are isogenic to NOTCH, we performed CRISPR/Cas9 LOF negative selection screens (using an sgRNA library enriched in druggable enzymes) to identify synthetic lethal interactors with LOF NOTCH mutations. Our CRISPR/Cas9 screen identified TRIM28 (Tripartite Motif Containing 28, or KAP1) as a highly significant synthetic lethal interactor with NOTCH1 or NOTCH2. We validated the synthetic lethal interaction between NOTCH1/2 and TRIM28 using both human and mouse NOTCH-isogenic cell lines. Interestingly, RNA-sequencing of NOTCH2-Mutant or NOTCH-WT SCLC cell lines after TRIM28 CRISPR inactivation showed robust increases in endogenous retroviral (ERV) expression and the MDA5/RIG-I/MAVS RNA-sensing machinery leading to hyperactivation of the TBK1/IRF3/7 innate immune signaling pathway and the pro-inflammatory cytokine CXCL10 selectively in cells with NOTCH inactivated. Notably, inactivation of TBK1 activity or JAK-STAT activity using specific small molecule inhibitors of TBK1 or JAK completely reversed the synthetic lethality between NOTCH and TRIM28 suggesting that the synthetic lethality phenotype is a consequence of hyperactivation of innate immune signaling, which is known to be cytotoxic. Together our findings uncover a novel vulnerability in SCLCs with LOF NOTCH mutations and perhaps other cancers with LOF NOTCH mutations and suggest a strategy that could also potentiate anti-tumor immunity by increasing innate immune signaling in tumor cells. Citation Format: Deli Hong, Matthew A. Booker, Sidrah Anjum, Yixiang Li, Tran C. Thai, Michelle Y. Wang, David A. Barbie, Michael Y. Tolstorukov, Jun Qi, Matthew G. Oser. Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH. [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 5727.

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. While these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (e.g., population frequency, functional and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation and importantly impacts patient care. Therefore, it’s essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium (CGC), and the Variant Interpretation for Cancer Consortium (VICC) used a consensus approach to develop a Standard Operating Procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification, and has been validated on 94 somatic variants in ten common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of a somatic variant.

Chromatin attains its three-dimensional (3D) conformation by establishing contacts between different noncontiguous regions. Sterile Alpha Motif (SAM)–mediated polymerization of the polyhomeotic (PH) protein regulates subnuclear clustering of Polycomb Repressive Complex 1 (PRC1) and chromatin topology. The mutations that perturb the ability of the PH to polymerize, disrupt long-range chromatin contacts, alter Hox gene expression, and lead to developmental defects. To understand the underlying mechanism, we combined the experiments and theory to investigate the effect of this SAM domain mutation on nucleosome occupancy and accessibility on a genome wide scale. Our data show that disruption of PH polymerization because of SAM domain mutation decreases nucleosome occupancy and alters accessibility. Polymer simulations investigating the interplay between distant chromatin contacts and nucleosome occupancy, both of which are regulated by PH polymerization, suggest that nucleosome density increases when contacts between different regions of chromatin are established. Taken together, it appears that SAM domain–mediated PH polymerization biomechanically regulates the organization of chromatin at multiple scales from nucleosomes to chromosomes and we suggest that higher order organization can have a top–down causation effect on nucleosome occupancy.

Some small cell lung cancers (SCLCs) are highly sensitive to inhibitors of the histone demethylase LSD1. LSD1 inhibitors are thought to induce their anti-proliferative effects by blocking neuroendocrine differentiation, but the mechanisms by which LSD1 controls the SCLC neuroendocrine phenotype are not well understood. To identify genes required for LSD1 inhibitor sensitivity in SCLC, we performed a positive selection genome-wide CRISPR/Cas9 loss of function screen and found that ZFP36L1, an mRNA-binding protein that destabilizes mRNAs, is required for LSD1 inhibitor sensitivity. LSD1 binds and represses ZFP36L1 and upon LSD1 inhibition, ZFP36L1 expression is restored, which is sufficient to block the SCLC neuroendocrine differentiation phenotype and induce a non-neuroendocrine “inflammatory” phenotype. Mechanistically, ZFP36L1 binds and destabilizes SOX2 and INSM1 mRNAs, two transcription factors that are required for SCLC neuroendocrine differentiation. This work identifies ZFP36L1 as an LSD1 target gene that controls the SCLC neuroendocrine phenotype and demonstrates that modulating mRNA stability of lineage transcription factors controls neuroendocrine to non-neuroendocrine plasticity.

Introduction STK11 and KEAP1 mutations (STK11 mutant [STK11MUT] and KEAP1MUT) are among the most often mutated genes in lung adenocarcinoma (LUAD). Although STK11MUT has been associated with resistance to programmed death-(ligand)1 (PD-[L]1) inhibition in KRASMUT LUAD, its impact on immunotherapy efficacy in KRAS wild-type (KRASWT) LUAD is currently unknown. Whether KEAP1MUT differentially affects outcomes to PD-(L)1 inhibition in KRASMUT and KRASWT LUAD is also unknown. Methods Clinicopathologic and genomic data were collected from September 2013 to September 2020 from patients with advanced LUAD at the Dana-Farber Cancer Institute/Massachusetts General Hospital cohort and the Memorial Sloan Kettering Cancer Center/MD Anderson Cancer Center cohort. Clinical outcomes to PD-(L)1 inhibition were analyzed according to KRAS, STK11, and KEAP1 mutation status in two independent cohorts. The Cancer Genome Atlas transcriptomic data were interrogated to identify differences in tumor gene expression and tumor immune cell subsets, respectively, according to KRAS/STK11 and KRAS/KEAP1 comutation status. Results In the combined cohort (Dana-Farber Cancer Institute/Massachusetts General Hospital + Memorial Sloan Kettering Cancer Center/MD Anderson Cancer Center) of 1261 patients (median age = 61 y [range: 22–92], 708 women [56.1%], 1065 smokers [84.4%]), KRAS mutations were detected in 536 cases (42.5%), and deleterious STK11 and KEAP1 mutations were found in 20.6% (260 of 1261) and 19.2% (231 of 1202) of assessable cases, respectively. In each independent cohort and in the combined cohort, STK11 and KEAP1 mutations were associated with significantly worse progression-free (STK11 hazard ratio [HR] = 2.04, p Conclusions STK11 and KEAP1 mutations confer worse outcomes to immunotherapy among patients with KRASMUT but not among KRASWT LUAD. Tumors harboring concurrent KRAS/STK11 and KRAS/KEAP1 mutations display distinct immune profiles in terms of gene expression and immune cell infiltration.

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2β, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2β arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2β also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2β loss. Mi-2β stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2β shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2β promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.

Neuron-glia interactions play a critical role in the regulation of synapse formation and circuit assembly. Here we demonstrate that canonical Sonic hedgehog (Shh) pathway signaling in cortical astrocytes acts to coordinate layer-specific synaptic connectivity. We show that the Shh receptor Ptch1 is expressed by cortical astrocytes during development and that Shh signaling is necessary and sufficient to promote the expression of genes involved in regulating synaptic development and layer-enriched astrocyte molecular identity. Loss of Shh in layer V neurons reduces astrocyte complexity and coverage by astrocytic processes in tripartite synapses; conversely, cell-autonomous activation of Shh signaling in astrocytes promotes cortical excitatory synapse formation. Furthermore, Shh-dependent genes Lrig1 and Sparc distinctively contribute to astrocyte morphology and synapse formation. Together, these results suggest that Shh secreted from deep-layer cortical neurons acts to specialize the molecular and functional features of astrocytes during development to shape circuit assembly and function.

Neuron-glia interactions play a critical role in the regulation of synapse formation and neuron specification. The cellular and molecular mechanisms by which neurons and astrocytes communicate and coordinate are not well understood. Here we demonstrate that canonical Sonic hedgehog (Shh) pathway signaling in cortical astrocytes acts to coordinate layer-specific synaptic connectivity and functional circuit development. We show that the Shh receptor Ptch1 is expressed by cortical astrocytes during development and that Shh signaling is necessary and sufficient to promote the expression of genes involved in regulating synaptic development and layer-specific astrocyte molecular identity. Loss of Shh in layer V neurons reduces astrocyte complexity and coverage by astrocytic processes in tripartite synapses, conversely, cell-autonomous activation of Shh signaling in astrocytes promotes cortical excitatory synapse formation. Furthermore, we determined that Shh-dependent genes Lrig1 and Sparc distinctively contribute to astrocyte morphology and synapse formation. Together, these results suggest that Shh secreted from deep layer cortical neurons acts to specialize the molecular and functional features of astrocytes during development to shape circuit assembly and function.

Polyethylene (PE) has emerged recently as a promising polymer for incorporation in wearable textiles owing to its high infrared transparency and tuneable visible opacity, which allows the human body to cool via thermal radiation, potentially saving energy on building refrigeration. Here, we show that single-material PE fabrics may offer a sustainable, high-performance alternative to conventional textiles, extending beyond radiative cooling. PE fabrics exhibit ultra-light weight, low material cost and recyclability. Industrial materials sustainability (Higg) index calculations predict a low environmental footprint for PE fabrics in the production phase. We engineered PE fibres, yarns and fabrics to achieve efficient water wicking and fast-drying performance which, combined with their excellent stain resistance, offer promise in reducing energy and water consumption as well as the environmental footprint of PE textiles in their use phase. Unlike previously explored nanoporous PE materials, the high-performance PE fabrics in this study are made from fibres melt spun and woven on standard equipment used by the textile industry worldwide and do not require any chemical coatings. We further demonstrate that these PE fibres can be dry coloured during fabrication, resulting in dramatic water savings without masking the PE molecular fingerprints scanned during the automated recycling process. The textile industry is one of the largest polluters. Here the authors show that polyethylene is a sustainable alternative textile with water wicking and fast-drying performance. The fabrication of polyethylene fabrics is compatible with standard equipment and could be dry-coloured, further reducing water consumption.

Brain metastases (BM) are a leading cause of cancer death and prognosis remains poor despite treatment advances at other sites. Models are central to therapeutic development, but few orthotopic patient-derived xenograft (PDX) models of BM exist. To represent diversity across BM types, we established a program to create orthotopic PDX at scale from all BM patients. To date BM were received from 100 patients and PDX attempted by direct brain injection (PDX, n=89) or injection of low passage patient-derived cell lines (PDCLX, n=11). We created 65 successful BM PDX from 13 cancers: 17 lung (55% take), 15 breast (68%), 6 melanoma (75%), 5 CNS lymphoma (83%), 3 gastrointestinal (75%), 2 esophageal (40%), 2 ovarian (67%), 1 sarcoma (100%), 1 laryngeal (100%), 1 prostate (100%), 1 pancreatic (100%), 1 uterine adenosarcoma (100%), and 1 yolk sac tumor (100%). Take rate was similar for models derived from patients with prior chemotherapy-only versus immune/targeted therapy-only (63 vs 58%). Fifteen patients had live tumor and matching PBMCs archived for modeling in vitro immunotherapy responses. Mean time to moribund among different cancer types ranged from 27 days (yolk sac tumor) to 177.5 days (ovarian). BM PDX had a favorable timeline for preclinical study (90% moribund at 180 days). All PDX retained high fidelity to the patient driver SNVs and copy aberrations, even at >P4. No significant differences noted by immunodeficient strain (SCID versus NSG) or injection site (orthotopic versus heterotopic). Explants from BM PDX were able to generate long-term cell lines (60%) or short-term cultures with qualitative concordance of model-to-patient responses to targeted therapy (Osimertinib, EGFRi) and immunotherapy (Pembrolizumab, PD1i). Genomic and clinical data were used to create the DFCI BM PDX cBioPortal for public release and models distribution will be available through the DFCI Center for Patient Derived Models.

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack. Significance: Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell–intrinsic type 1 interferon response. This article is highlighted in the In This Issue feature, p. 1861