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During prolonged exposure to antigens, such as chronic viral infections, sustained T cell receptor (TCR) signaling can result in T cell exhaustion mediated in part by expression of Programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. Here, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells, suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation, as well as increased surface levels of PD-1 during acute but not chronic infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute but not chronic LCMV infection. Loss of DNA methylation at Pdcd1’s promoter proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection where PD-1 expression levels return to near base line, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1 expressing tumor infiltrating LSD1-deficient CD8 T cells was greater than wild-type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and the regulation of a critical T cell checkpoint gene.Key PointsLSD1 suppress PD-1 expression following acute infection or transient induction.Blimp-1 binding to the Pdcd1 locus is required to recruit LSD1.LSD1 is required to fully remethylate the PD-1 proximal promoter region.

A major driver of multiple myeloma (MM) is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employed an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in MM to inform precision medicine strategies. Unbiased phosphoproteomics initially revealed differential activation of kinases across MM cell lines and that sensitivity to mammalian target of rapamycin (mTOR) inhibition may be particularly dependent on mTOR kinase baseline activity. We further noted differential activity of immediate downstream effectors of Ras as a function of cell line genotype. We extended these observations to patient transcriptome data in the Multiple Myeloma Research Foundation CoMMpass study. A machine-learning–based classifier identified surprisingly divergent transcriptional outputs between NRAS- and KRAS-mutated tumors. Genetic dependency and gene expression analysis revealed mutated Ras as a selective vulnerability, but not other MAPK pathway genes. Transcriptional analysis further suggested that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs wild-type RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes, whereas RAS mutations overall carry no survival impact. We further developed an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. These results will lead to improved stratification of MM patients in precision medicine trials while also revealing unexplored modes of Ras biology in MM.

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed ‘high-risk’. To better understand and identify high-risk myeloma, we analyzed the translocation landscape of 826 newly-diagnosed patients by whole genome sequencing as part of the CoMMpass study. Translocations at the IgL locus were present in 10% of myeloma patients, and corresponded with poor prognosis. Importantly, 70% of IgL translocations co-occurred with hyperdiploid disease, a marker of standard risk, which is routinely diagnosed clinically whereas IgL-translocations are not. Thus, it is likely that the majority of IgL-translocated myeloma is being misclassified. The IgL enhancer is among the strongest in myeloma cells, indicating it can robustly drive oncogene expression when translocated. Consistent with this, IgL-translocated patients failed to benefit from immunomodulatory imide drugs (IMiDs), which target the lymphocyte-specific transcription factor Ikaros. These data implicate the IgL enhancer as resistant to IMiD-inhibition, and when translocated, as a driver of poor prognosis.

Monoallelic point mutations of the NADP+-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1R132H allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1R132H/WT mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1R132H/WT-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1R132H/WT knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1R132H/WT mutants in driving epigenetic instability in human cancer cells.