Sodium-glucose cotransporter-2 inhibitors for hypergycemia in phosphoinositide 3-kinase pathway inhibition.

PURPOSE: Phosphoinositide 3-kinase (PI3K) inhibition is used for the treatment of certain cancers, but can cause profound hyperglycemia and insulin resistance, for which sodium-glucose cotransporter-2 (SGLT2) inhibitors have been proposed as a preferred therapy. The objective of this research is to assess the effectiveness and safety of SGLT2 inhibitors for hyperglycemia in PI3K inhibition. METHODS: We conducted a single-center retrospective review of adults initiating the PI3K inhibitor alpelisib. Exposure to different antidiabetic drugs and adverse events including diabetic ketoacidosis (DKA) were assessed through chart review. Plasma and point-of-care blood glucoses were extracted from the electronic medical record. Change in serum glucose and the rate of DKA on SGLT2 inhibitor versus other antidiabetic drugs were examined as co-primary outcomes. RESULTS: We identified 103 patients meeting eligibility criteria with median follow-up of 92 days after starting alpelisib. When SGLT2 inhibitors were used to treat hyperglycemia, they were associated with a decrease in mean random glucose by -46 mg/dL (95% CI - 77 to - 15) in adjusted linear modeling. Five cases of DKA were identified, two occurring in patients on alpelisib plus SGLT2 inhibitor. Estimated incidence of DKA was: alpelisib plus SGLT2 inhibitor, 48 DKA cases per 100 patient-years (95% CI 6, 171); alpelisib with non-SGLT2 inhibitor antidiabetic drugs, 15 (95% CI 2, 53); alpelisib only, 4 (95% CI 0.1, 22). CONCLUSIONS: SGLT2 inhibitors are effective treatments for hyperglycemia in the setting of PI3K inhibition.

Induction of sarcomas by mutant IDH2.

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.

Critical role for PI3-kinase in regulating the use of proteins as an amino acid source.

Ras-transformed cells can grow in amino acid-poor environments by recovering amino acids through macropinocytosis and lysosomal catabolism of extracellular proteins. However, when studying nontransformed fibroblasts, we found that Ras GTPases are dispensable for growth-factor-stimulated macropinocytosis and lysosomal catabolism of extracellular proteins. Instead, we establish a critical role for phosphatidylinositol 3-kinase (PI3-kinase) signaling in cell proliferation that is supported by protein macropinocytosis. Downstream of PI3-kinase, distinct effectors have opposing roles in regulating uptake and catabolism of extracellular proteins. Rac1 and PLC are required for nutritional use of extracellular proteins. In contrast, Akt suppresses lysosomal catabolism of ingested proteins when free amino acids are abundant. The interplay between these pathways allows cells with oncogenic PIK3CA mutations or PTEN deletion to grow using diverse amino acid sources. Thus, the prevalence of PI3-kinase and PTEN mutations in cancer may result in part because they allow cells to cope with fluctuating nutrient availability.

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3' untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase-RAC-alpha serine/threonine-protein kinase-mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability.

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

HER2 Antibody-Drug Conjugates Are Active against Desmoplastic Small Round Cell Tumor.

PURPOSE: Desmoplastic small round cell tumor (DSRCT) is a rare but highly aggressive soft tissue sarcoma that arises in the abdominopelvic cavity of young males. Since the discovery of EWSR1::WT1 fusion as the driver of DSRCT, no actionable genomic alterations have been identified, limiting disease management to a combination of surgery, chemotherapy, and radiation, with very poor outcomes. Herein, we evaluated ERBB2/HER2 expression in DSRCT as a therapeutic target. EXPERIMENTAL DESIGN: ERBB2/HER2 expression was assessed in clinical samples and patient-derived xenografts (PDX) using RNA sequencing, RT-qPCR, and a newly developed HER2 IHC assay (clone 29D8). Responses to HER2 antibody-drug conjugates (ADC)-trastuzumab deruxtecan (T-DXd) and trastuzumab emtansine-were evaluated in DSRCT PDX, cell line, and organoid models. Drug internalization was demonstrated by live microscopy. Apoptosis was evaluated by Western blotting and caspase activity assays. RESULTS: ERBB2/HER2 was detectable in DSRCT samples from patients and PDXs, with higher sensitivity RNA assays and improved IHC detectability using clone 29D8. Treatment of ERBB2/HER2-expressing DSRCT PDX, cell line, and organoid models with T-DXd or trastuzumab emtansine resulted in tumor regression. This therapeutic response was long-lasting in T-DXd-treated xenografts and was mediated by rapid HER2 ADC complex internalization and cytotoxicity, triggering p53-mediated apoptosis and growth arrest. Xenograft regression was associated with bystander payload effects triggering global tumor niche responses proportional to HER2 status. CONCLUSIONS: ERBB2/HER2 is a therapeutic target in DSRCT. HER2 ADCs may represent novel options for managing this exceptionally aggressive sarcoma, possibly fulfilling an urgent and historically unmet need for more effective clinical therapy.

Sodium-Glucose Cotransporter-2 Inhibitors for Hyperglycemia in Phosphoinositide 3-kinase Pathway Inhibition.

Purpose Phosphoinositide 3-kinase (PI3K) inhibition is used for the treatment of certain cancers, but can cause profound hyperglycemia and insulin resistance, for which sodium-glucose cotransporter-2 (SGLT2) inhibitors have been proposed as a preferred therapy. The objective of this research is to assess the effectiveness and safety of SGLT2 inhibitors for hyperglycemia in PI3K inhibition. Methods We conducted a single-center retrospective review of adults initiating the PI3k inhibitor alpelisib. Exposure to different antidiabetic drugs and adverse events including diabetic ketoacidosis (DKA) were assessed through chart review. Plasma and point-of-care blood glucoses were extracted from the electronic medical record. Change in serum glucose and the rate of DKA on SGLT2 inhibitor versus other antidiabetic drugs were examined as co-primary outcomes. Results We identified 103 patients meeting eligibility criteria with median follow-up of 85 days after starting alpelisib. When SGLT2 inhibitors were used to treat hyperglycemia, they were associated with a decrease in mean random glucose by -54 mg/dL (95% CI -99 to -8) in adjusted linear modeling. Five cases of DKA were identified, two occurring in patients on alpelisib plus SGLT2 inhibitor. Estimated incidence of DKA was: alpelisib plus SGLT2 inhibitor, 24 DKA cases per 100 patient-years (95% CI 6, 80); alpelisib with non-SGLT2 inhibitor antidiabetic drugs, 7 (95% CI 0.1, 34); alpelisib only, 4 (95% CI 0.1, 21). Conclusions SGLT2 inhibitors are effective treatments for hyperglycemia in the setting of PI3K inhibition, but given possible adverse events, SGLT2 inhibitors should be used with caution.

Hypoxia Induces Production of L-2-Hydroxyglutarate.

Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the "oncometabolite" D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of α-ketoglutarate (α-KG)-dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D-enantiomer or the L-enantiomer. Although cancer-associated IDH1/2 mutants produce D-2HG, biochemical studies have demonstrated that L-2HG also functions as a potent inhibitor of α-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of α-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage primarily by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3).