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Purpose: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). Experimental Design: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. Results: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. Conclusions: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648

PURPOSE Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship with NF2 loss. Given the predominance of NF2 mutations in meningiomas, we evaluated the efficacy of GSK2256098, a FAK inhibitor, as part of the first genomically driven phase II study in recurrent or progressive grade 1-3 meningiomas. PATIENTS AND METHODS Eligible patients whose tumors screened positively for NF2 mutations were treated with GSK2256098, 750 mg orally twice daily, until progressive disease. Efficacy was evaluated using two coprimary end points: progression-free survival at 6 months (PFS6) and response rate by Macdonald criteria, where PFS6 was evaluated separately within grade-based subgroups: grade 1 versus 2/3 meningiomas. Per study design, the FAK inhibitor would be considered promising in this patient population if either end point met the corresponding decision criteria for efficacy. RESULTS Of 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients with NF2 mutations were enrolled and treated: 12 grade 1 and 24 grade 2/3 patients. Across all grades, one patient had a partial response and 24 had stable disease as their best response to treatment. In grade 1 patients, the observed PFS6 rate was 83% (10/12 patients; 95% CI, 52 to 98). In grade 2/3 patients, the observed PFS6 rate was 33% (8/24 patients; 95% CI, 16 to 55). The study met the PFS6 efficacy end point both for the grade 1 and the grade 2/3 cohorts. Treatment was well tolerated; seven patients had a maximum grade 3 adverse event that was at least possibly related to treatment with no grade 4 or 5 events. CONCLUSION GSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive NF2-mutated meningiomas, compared with historical controls. The criteria for promising activity were met, and FAK inhibition warrants further evaluation for this patient population.

Brain metastases are the most common brain malignancy. This review discusses the studies presented at the third annual meeting of the Melanoma Research Foundation in the context of other recent reports on the biology and treatment of melanoma brain metastases (MBM). Although symptomatic MBM patients were historically excluded from immunotherapy trials, efforts from clinicians and patient advocates have resulted in more inclusive and even dedicated clinical trials for MBM patients. The results of checkpoint inhibitor trials were discussed in conversation with current standards of care for MBM patients, including steroids, radiotherapy, and targeted therapy. Advances in the basic scientific understanding of MBM, including the role of astrocytes and metabolic adaptations to the brain microenvironment, are exposing new vulnerabilities which could be exploited for therapeutic purposes. Technical advances including single-cell omics and multiplex imaging are expanding our understanding of the MBM ecosystem and its response to therapy. This unprecedented level of spatial and temporal resolution is expected to dramatically advance the field in the coming years and render novel treatment approaches that might improve MBM patient outcomes.

Supplementary Figures and Figure Legends

Table S2. Liquid Chromatography and Mass Spectrometry Measurement of 2-hydroxyglutarate in the Conditioned Media of mIDH1 GCCs untreated, treated with vehicle (DMSO), or mIDH1 inhibitor (AGI-5198 or DS1001b)

Purpose:Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR).Experimental Design:We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP.Results:Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells.Conclusions:These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma.See related commentary by Sachdev et al., p. 1648

Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Supplementary Figure from Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition

Supplementary Data from Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC.Significance:Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

Summary of clinically informative (TARGET) genes.

Figure S1. A compound screen identifies Volasertib as potently cytotoxic to MSH6-deficient glioblastoma cells. Figure S2. The effects of temozolomide and PLK1 inhibitors on glioblastoma cells with and without MSH6 knockdown. Figure S3. Combination of Volasertib and temozolomide on glioblastoma cells with and without MSH6 knockdown. Figure S4. PLK1 inhibitor effects in orthotopic glioblastoma models. Figure S5. In vitro PLK1 inhibitor effects on normal astrocytes and glioblastoma cells. Figure S6. Myc expression and PLK1 inhibitor effects.

Supplementary Methods, Figures S1 - S20. Figure S1. Branched evolution leads to tissue-sampling bias in primary tumor samples. Figure S2. Results of ABSOLUTE on samples from patient 418. Figure S3. 2D Bayesian clustering analysis of point-mutation CCF distributions in case 418. Figure S4. Bayesian clustering of private point-mutation CCF distributions in all sequenced tissue samples from case 418. Figure S5. Genetic alterations supporting phylogeny construction in case 418. Figure S6. Phylogenetic tree for case 418 Figure S7. Evolutionary relationships between primary tumor-samples and brain metastasis samples Figure S8. Detection of homozygous deletion in CDKN2A in the brain metastasis of case 24. Figure S9. Amplification of FGFR1 and MYC detected in the brain metastasis of case 331. Figure S10. Additional alterations under investigation for association with various targeted therapies. Figure S11. Power for paired-detection of somatic mutations. Figure S12. Amplification of CCNE1 detected in the brain metastasis of case 314. Figure S13. Amplification of EGFR detected in the brain metastasis of case 314. Figure S14. Amplification of MYC detected in the brain metastasis of case 308. Figure S15. Amplification of MYC detected in the brain metastasis of case 138. Figure S16. Amplifications of CDK6 and MET detected in the brain metastasis of case 138. Figure S17. Amplifications of CCNE1 and AKT2 detected in the brain metastasis of case 138. Figure S18. Amplification of EGFR detected in a regional lymph node from case 296. Figure S19. Power for somatic mutation detection in 86 matched primary-tumor and brain-metastasis samples. Figure S20. Calling of amplifications in primary-tumor samples and paired brain metastases.

NAD+ is an essential cofactor metabolite and is the currency of metabolic transactions critical for cell survival. Depending on tissue context and genotype, cancer cells have unique dependencies on NAD+ metabolic pathways. PARPs catalyze oligomerization of NAD+ monomers into PAR chains during cellular response to alkylating chemotherapeutics, including procarbazine or temozolomide. Here we find that, in endogenous IDH1-mutant tumor models, alkylator-induced cytotoxicity is markedly augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme PAR glycohydrolase (PARG). Both in vitro and in vivo, we observe that concurrent alkylator and PARG inhibition depletes freely available NAD+ by preventing PAR breakdown, resulting in NAD+ sequestration and collapse of metabolic homeostasis. This effect reversed with NAD+ rescue supplementation, confirming the mechanistic basis of cytotoxicity. Thus, alkylating chemotherapy exposes a genotype-specific metabolic weakness in tumor cells that can be exploited by PARG inactivation.Significance:Oncogenic mutations in the isocitrate dehydrogenase genes IDH1 or IDH2 initiate diffuse gliomas of younger adulthood. Strategies to maximize the effectiveness of chemotherapy in these tumors are needed. We discover alkylating chemotherapy and concurrent PARG inhibition exploits an intrinsic metabolic weakness within these cancer cells to provide genotype-specific benefit.See related commentary by Pirozzi and Yan, p. 1629.This article is highlighted in the In This Issue feature, p. 1611

Supplementary File 1. Power for detection of somatic point mutations in coding exons of TARGET genes. The description for the plots in Supplementary File 1 can be found in the Legend of Supplementary Figure S1.

Supplementary File 3. Detailed plots of the data used for phylogenetic inference in each case. The description for the plots in Supplementary File 3 can be found in the Legend of Supplementary Figure S13-S17.

shRNA and Control shRNA Cell Lines, Molecular Analyses, 2-HG Quantitation, Western blot analysis, Immunohistochemistry, TUNEL assay, In Vivo Studies

Supplementary File 2. Detailed plots of SCNA calls in TARGET genes. The description for the plots in Supplementary File 2 can be found in the Legend of Supplementary Figure S3.

Supplementary Figure 1-5, Supplementary Tables 1-3

Mismatch repair (MMR) deficiency through MSH6 inactivation has been identified in up to 30% of recurrent high-grade gliomas, and represents a key molecular mechanism underlying the acquired resistance to the alkylating agent temozolomide (TMZ). To develop a therapeutic strategy that could be effective in these TMZ-refractory gliomas, we first screened 13 DNA damage response modulators for their ability to suppress viability of MSH6-inactivated, TMZ-resistant glioma cells. We identified a PLK1 selective inhibitor, Volasertib, as the most potent in inhibiting proliferation of glioblastoma cells. PLK1 inhibition induced mitotic catastrophe, G2–M cell-cycle arrest, and DNA damage, leading to caspase-mediated apoptosis in glioblastoma cells. Importantly, therapeutic effects of PLK1 inhibitors were not influenced by MSH6 knockdown, indicating that their action is independent of MMR status of the cells. Systemic treatment with Volasertib potently inhibited tumor growth in an MMR-deficient, TMZ-resistant glioblastoma xenograft model. Further in vitro testing in established and patient-derived cell line panels revealed an association of PLK1 inhibitor efficacy with cellular Myc expression status. We found that cells with deregulated Myc are vulnerable to PLK1 inhibition, as Myc overexpression sensitizes, whereas its silencing desensitizes, glioblastoma cells to PLK1 inhibitors. This discovery is clinically relevant as glioma progression post-TMZ treatment is frequently accompanied by MYC genomic amplification and/or pathway activation. In conclusion, PLK inhibitor represents a novel therapeutic option for recurrent gliomas, including those TMZ-resistant from MMR deficiency. Genomic MYC alteration may serve as a biomarker for PLK inhibitor sensitivity, as Myc-driven tumors demonstrated pronounced responses.

Supplementary Methods

PDF file - 102KB, Supplementary Figure S6. Sphere formation assay of MGG152 expressing N-MYC shRNA.

List of differentially present metabolites. The table lists fold change value of metabolites differentially detected in CAF co-cultures compared to separately cultured cells in at least one of the cell lines. Bold font indicates statistically significant (p

XRCC1 inactivation does not restore the sensitivity to TMZ in MSH6-deficient cells

Supplementary Table 2. Relationship of NAMPT inhibitor sensitivity, MYC/MYCN amplification, and Myc/N-Myc protein expression status in glioblastoma tumorsphere lines

Supplementary Figures S1 and S2, new figures

''Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.''

PDF file - 266KB, Supplementary Figure S5. Maintenance of genotype upon serial xenotransplantation.

Supplementary Data from HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma

Table S1 - List of antibodies used

Supplementary Figure from HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma

'Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.'

Supplementary Figure S1. Nampt inhibitor effects in glioblastoma tumorsphere cells. Supplementary Figure S2. Characterization of Nampt inhibitor effects in MYC/MYCN driven glioblastoma cells. Supplementary Figure S3. Characterization of NAD+-synthesis pathways in glioblastoma cells. Supplementary Figure S4. Nampt inhibitors induce apoptosis and disrupt intracellular metabolism pathways in MYC/MYCN amplified glioblastoma cells. Supplementary Figure S5. Fluorescent in situ hybridization (FISH) for MYC and MYCN in standard cancer cell lines. Supplementary Figure S6. Effect of Nampt inhibitors in Myc-driven standard cancer cell lines. Supplementary Figure S7. In vivo efficacy of Nampt inhibitor in MYC amplified H1975 xenografts. Supplementary Figure S8. NAMPT inhibitors were well tolerated in SCID mice bearing flank or orthotopic xenografts.

PDF file - 404KB, Supplementary Figure S2. IDH1-mutant glioma orthotopic xenografts recapitulate histopathological features of the respective patient tumors.