Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH−, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. Implications: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with an estimated 1.6 million deaths each year. Non-small cell lung cancer (NSCLC) is with 85% by far the most common subtype of lung cancer, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common with the worst overall survival for KRAS mutant adenocarcinoma patients. We have established a murine model of lung cancer, wherein expression of oncogenic Kras can be controlled genetically, allowing activation of oncogenic KrasG12D (Kras*) to initiate tumor growth, tumor eradication upon Kras* depletion and re-activation as a means to model relapse. Oncogenic Kras depletion (deactivation) has previously been reported to result in tumor cell apoptosis even in the presence of tumor suppressor loss. However, the mechanisms of apoptosis, the role of the immune system on these changes, and the mechanisms allowing some tumor cells to escape apoptosis, which typically results in tumor relapse, are unknown. Here, we interrogated the immune response in mediating tumor regression and relapse using this genetically engineered models. Multiplex immunohistochemistry as well as CyTOF provided insight into the changes in immune contexture upon Kras* depletion in mice haploinsufficient for tumor suppressor p53 or mutant for p53 (R172H). Interestingly, total number of T cells including cytotoxic T cells (CTLs) was elevated in lung tumors from p53 mutant mice supporting findings of heightened immune activation and overall response to immune therapy with an increased mutational burden. Kras* inactivation and thus inhibition of MAPK signaling resulted in an overall decrease in abundance of CTLs and antigen presenting cells (APC) as well as engagement of CTL with tumor cells and APCs indicating a decrease in immune presence likely due to proceeding tumor cell kill and immune recruitment. However, intracellular distance of CTL with tumor cells indicated active tumor cell kill of the CTLs to eradicate remaining tumor cells. In summary, these findings support recent observation of increased immune activation in tumors with higher mutational load as well as changes mediated by inhibition of MAPK signaling which both maybe harnessed for enhancing future immunotherapies. Citation Format: Nina Steele, Kristena Y. Abdelmalak, Sarah F. Ferris, Jennifer M. Lee, Carlos Espinoza, Yaqing Zhang, Sundaresh Ram, Craig Galban, Nithya Ramnath, Timothy L. Frankel, Marina Pasca di Magliano, Stefanie Galbán. Oncogenic Kras-mediated regulation of the tumor microenvironment in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3855.

<p>Supplemental Figure S1. FACS analysis of Aldefluor stained SU-DIPG XIII cells. Supplemental Figure S2: Downstream signaling changes by inhibition of MAPK and PI3K/mTOR in DIPG cells. Supplemental Figure S3. Bru-sequencing transcriptome analysis identifies gene sets regulated by MAPK and PI3K/mTOR inhibition in ALDH positive SU-DIPG XIII cells. Supplemental Figure S4. A-C. Heatmaps of Bru-seq RPKM values in single agent treated (901 or GSK) ALDH+ SU-DIPG XII cells highlight treatment effects on "stemness" and DDR genes. Supplemental Figure S5: A.-B. Illustrative result of BOILED-Egg predictive model for GSK-458 and GDC-0084, respectively. Supplemental Figure S6: A-C. SU-DIPG-XIII cells were Aldefluor stained and sorted into an ALDH+ and ALDH- population. Supplemental Figure S7: A. Aldefluor stain and FACS of SU-DIPG XIII cells. B. ALDH+ cells were treated with 50 nM 901+GSK each and 24 hrs later Aldefluor re-stained and analyzed by flow cytometry. Table S1. Characteristics of Patient-Derived Diffuse Intrinsic Pontine Glioma (DIPG) Cells. Table S2. Selected Genes Upregulated in ALDH+ DIPG cells Table S3. Selected Metabolome Genes Upregulated in ALDH+ DIPG Cells. Table S4. RT-qPCR primer sequences.</p>

<p>Supplemental Figure S1. FACS analysis of Aldefluor stained SU-DIPG XIII cells. Supplemental Figure S2: Downstream signaling changes by inhibition of MAPK and PI3K/mTOR in DIPG cells. Supplemental Figure S3. Bru-sequencing transcriptome analysis identifies gene sets regulated by MAPK and PI3K/mTOR inhibition in ALDH positive SU-DIPG XIII cells. Supplemental Figure S4. A-C. Heatmaps of Bru-seq RPKM values in single agent treated (901 or GSK) ALDH+ SU-DIPG XII cells highlight treatment effects on "stemness" and DDR genes. Supplemental Figure S5: A.-B. Illustrative result of BOILED-Egg predictive model for GSK-458 and GDC-0084, respectively. Supplemental Figure S6: A-C. SU-DIPG-XIII cells were Aldefluor stained and sorted into an ALDH+ and ALDH- population. Supplemental Figure S7: A. Aldefluor stain and FACS of SU-DIPG XIII cells. B. ALDH+ cells were treated with 50 nM 901+GSK each and 24 hrs later Aldefluor re-stained and analyzed by flow cytometry. Table S1. Characteristics of Patient-Derived Diffuse Intrinsic Pontine Glioma (DIPG) Cells. Table S2. Selected Genes Upregulated in ALDH+ DIPG cells Table S3. Selected Metabolome Genes Upregulated in ALDH+ DIPG Cells. Table S4. RT-qPCR primer sequences.</p>

<p>Combined pathway analyses of sequencing data</p>