Isocitrate Dehydrogenase Mutation Research Articles

Development and validation of clinical-radiomics analysis for preoperative prediction of IDH mutation status and WHO grade in diffuse gliomas: a consecutive L-[methyl-11C] methionine cohort study with two PET scanners.

Determination of isocitrate dehydrogenase (IDH) genotype is crucial in the stratification of diagnosis and prognostication in diffuse gliomas. We sought to build and validate radiomics models and clinical features incorporated nomogram for preoperative prediction of IDH mutation status and WHO grade of diffuse gliomas with L-[methyl-11C] methionine ([11C]MET) PET/CT imaging according to the 2016 WHO classification of tumors of the central nervous system. Consecutive 178 preoperative [11C]MET PET/CT images were retrospectively studied for radiomics analysis. One hundred six patients from PET scanner 1 were used as training dataset, and 72 patients from PET scanner 2 were used for validation dataset. [11C]MET PET and integrated CT radiomics features were extracted, respectively; three independent predictive models were built based on PET features, CT features, and combined PET/CT features, respectively. The SelectKBest method, Spearman correlation analysis, Least Absolute Shrinkage and Selection Operator (LASSO) regression, and machine learning algorithms were applied for feature selection and model building. After filtering the satisfactory predictive model, key clinical features were incorporated for the nomogram establishment. The combined [11C]MET PET/CT radiomics model, which consisted of four PET features and eight integrated CT features, was significantly associated with IDH genotype (p < 0.0001 for both training and validation datasets). Nomogram based on the [11C]MET PET/CT radiomics score, patients' age, and dichotomous tumor location status showed satisfactory discrimination capacity, and the AUC was 0.880 (95% CI, 0.726-0.998) in the training dataset and 0.866 (95% CI, 0.777-0.956) in the validation dataset. In IDH stratified WHO grade prediction, the final radiomics model consists of four PET features and two CT features had reasonable and stable differential efficacy of WHO grade II and III patients from grade IV patients in IDH-wildtype patients, and the AUC was 0.820 (95% CI, 0.541-1.000) in the training dataset and 0.766 (95% CI, 0.612-0.921) in the validation dataset. [11C]MET PET radiomics features could benefit non-invasive IDH genotype prediction, and integrated CT radiomics features could enhance the efficacy. Radiomics and clinical features incorporation could establish satisfactory nomogram for clinical application. This non-invasive predictive investigation based on our consecutive cohort from two PET scanners could provide the perspective to observe the differential efficacy and the stability of radiomics-based investigation in untreated diffuse gliomas.

A biosensor for D-2-hydroxyglutarate in frozen sections and intraoperative assessment of IDH mutation status

The oncometabolite D-2-hydroxyglutarate (D-2-HG) has emerged as a valuable biomarker in tumors with isocitrate dehydrogenase (IDH) mutations. Efficient detection methods are required and rapid intraoperative determination of D-2-HG remains a huge challenge. Herein, D-2-HG dehydrogenase from Achromobacter xylosoxidans (AX-D2HGDH) was found to have high substrate specificity. AX-D2HGDH dehydrogenizes D-2-HG and reduces flavin adenine dinucleotide (FAD) bound to the enzyme. Interestingly, the dye resazurin can be taken as another substrate to restore FAD. AX-D2HGDH thus catalyzes a bisubstrate and biproduct reaction: the dehydrogenation of D-2-HG to 2-ketoglutarate and simultaneous reduction of non-fluorescent resazurin to highly fluorescent resorufin. According to steady-state analysis, a ping-pong bi-bi mechanism has been concluded. The Km values for resazurin and D-2-HG were determined as 0.56 μM and 10.93 μM, respectively, suggesting high affinity to both substrates. On the basis, taking AX-D2HGDH and resazurin as recognition and fluorescence transducing element, a D-2-HG biosensor (HGAXR) has been constructed. HGAXR exhibits high sensitivity, accuracy and specificity for D-2-HG in different biological samples. With the aid of HGAXR and the matched low-cost palm-size detecting device, D-2-HG levels in frozen sections of resected brain tumor tissues can be measured in a direct, simple and accurate manner with a fast detection (1–3 min). As the technique of frozen section is familiar to surgeons and pathologists, HGAXR and the portable device can be easily integrated into the current workflow, having potential to provide rapid intraoperative pathology for IDH mutation status and guide decision-making during surgery.

The development of a hiPSC-based platform to identify tissue-dependencies of IDH1 R132H

The application of patient-derived (PD) in vitro tumor models represents the classical strategy for clinical translational oncology research. Using these cellular heterogeneous cultures for the isolation of cancer stem cells (CSCs), suggested to be the main driver for disease malignancy, relies on the use of surrogate biomarkers or is based on CSC-enriching culture conditions. However, the ability of those strategies to exclusively and efficiently enrich for CSC pool has been questioned. Here we present an alternative in vitro CSC model based on the oncogenic transformation of single clone-derived human induced pluripotent stem cells (hiPSC). Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) and codon R175 of p53 are commonly occurring molecular features of different tumors and were selected for our transformation strategy. By choosing p53 mutant glial tumors as our model disease, we show that in vitro therapy discovery tests on IDH1-engineered synthetic CSCs (sCSCs) can identify kinases-targeting chemotherapeutics that preferentially target tumor cells expressing corresponding genetic alteration. In contrast, neural stem cells (NSCs) derived from the IDH1R132H overexpressing hiPSCs increase their resistance to the tested interventions indicating glial–to-neural tissue-dependent differences of IDH1R132H. Taken together, we provide proof for the potential of our sCSC technology as a potent addition to biomarker-driven drug development projects or studies on tumor therapy resistance. Moreover, follow-up projects such as comparing in vitro drug sensitivity profiles of hiPSC-derived tissue progenitors of different lineages, might help to understand a variety of tissue-related functions of IDH1 mutations.

Survival Analysis and Correlates with Molecular Epidemiology: 10-Year Retrospective Series of High-Grade Glioma in Pakistan.

High-grade gliomas are malignant, recurring primary central nervous system (CNS) tumors requiring extensive postoperative chemotherapy and radiation treatment. Isocitrate dehydrogenase (IDH), 1p19q, and ATRX mutations significantly influence survival and response to chemotherapy, as seen in many extensive studies from the Global North. This study aims to report data from the local region regarding progression-free survival and overall survival in light of molecular characteristics. A 10-year retrospective series was conducted at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, with 285 patients presenting from 2008 to 2018. Prospective follow-up data was collected, and complete molecular profiles were available for patients presenting from 2010 onwards. Survival analysis was conducted through the Kaplan-Meier method, with log-rank reported. 70.53% (201) of patients were male, with a mean age at diagnosis of 43.33 ± 15.1 years. 265 patients within the cohort completed postoperative radiotherapy, while 141 patients underwent chemotherapy (procarbazine, lomustine, and vincristine, or temozolomide). Mean survival, in months, within the cohort was as follows: glioblastoma (14.1), anaplastic astrocytoma (27.5), and anaplastic oligodendroglioma (39.8). Survival curves showed a lower survival for IDH wild-type (P < 0.0001), ATRX mutated (P = 0.029), and 1p19q non-deleted (P = 0.008) tumors from Pakistan. Our findings quantified long-term survival outcomes for high-grade glioma from Pakistan, analyzing the various treatment patterns. Of particular importance, molecular sub-classification significantly predicted survival outcomes for IDH, ATRX, and 1p19 co-deletion mutations. Expanding brain tumor epidemiology will benefit assessing the efficacy of regional oncological centers and establishing standards of care.

Abstract C036: Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q

Abstract According to the National Cancer Institute, there were 25,050 new brain cancer cases in 2022, representing 1.3% of all cancer incidents in the United States. In the same year, the estimated death was 18,280 cases, representing 3.0% of all cancer deaths. Relative survival statistics between 2017 and 2018 showed that only 32.5% of patients would survive five years or more. Disparities in cancer incidence and outcomes, including brain cancers, can be due to biological, socioeconomic, and care access factors that often have roots in structural racism. Research shows lower three years survival rates of glioblastoma in the African American population by 28.8% compared to the Asian Pacific Islanders at 36.6% and Hispanic at 32.3%. Mutant isocitrate dehydrogenase 1 (IDH1) can lead to brain cancer, including &amp;gt; 80% of lower-grade gliomas and secondary glioblastomas. Mutant IDH1 catalyzes the NADPH-dependent conversion of α-ketoglutarate to D2-hydroxyglutarate (D2HG), an oncometabolite linked to many cancer types. IDH1 mutation occurs primarily at residue 132, and our lab has shown that the catalytic activity differs based on the amino acid substituted at that location. There is a lack of information describing the relation of D2HG produced to cancer cells’ phenotype severity. Understanding IDH1 mutation will clarify cancer progression, patients’ prognosis, and treatment responses. Here, we hypothesize that the toxic effect of D2HG will impair the viability of cells expressing mutant IDH1 and will cause a reduction in ROS and NADPH cellular levels. This work examines D2HG production efficiency in vivo and in vitro and the metabolic consequences of this oncometabolite. We created cell lines stably overexpressing IDH1-R132Q and IDH1-R132H to conduct this work. Our results showed higher levels of D2HG in cells expressing IDH1-R132Q compared to DH1-wild type and IDH1-R132H, supportive of our previous finding that IDH1-R132Q mutation has higher catalytic activities (higher efficiency in producing mutant IDH1). These higher D2HG levels caused cells to proliferate and migrate at lower rates. D2HG interferes with DNA demethylation by inhibiting the TET enzyme, and we observed higher levels of histone protein expression in IDH1-R132Q compared to the other modified cells. In in vivo studies, we identified elevated D2HG levels in mouse xenografts expressing IDH1-R132Q that affected tumor growth. Citation Format: Mowaffaq A.A. Adam, Mikella Robinson, An Hoang, Grace A. Wells, David A. Scott, Olga Zagnitko, Carrie House, Christal D. Sohl. Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C036.

Abstract B040: NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive malignancy with mutations in Isocitrate dehydrogenase (IDH) identified in approximately 15% of patients. Neomorphic mutations in IDH 1/2 result in accumulation of the oncometabolite 2-hydroxyglutarate (2G), which has been implicated in tumor progression via inhibitory effects on alpha ketoglutarate. Moreover, the mutant IDH dependent accumulation of 2HG results in homologous recombination deficiency (HRD) and renders these cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. NCI 10129 evaluated the efficacy of olaparib for IDH mutated solid tumors, and here we present the CCA cohort. Methods: NCI 10129 is a 3 arm, open label phase 2 clinical trial performed in the NCI National Clinical Trials Network evaluating olaparib 300 mg twice daily for IDH mutated solid tumors refractory to standard treatment. Patients with CCA were enrolled in 2 cohorts 1) IDHi pre-treated CCA and 2) IDHi untreated CCA. The primary endpoint was overall response rate (ORR) and secondary endpoints includes progression-free survival (PFS), and overall survival (OS). An exploratory analysis measured the impact of circulating 2HG levels, circulating tumor DNA, and tumor genomics on patient outcomes. Results: Thirty patients with CCA enrolled on NCI10129 (15 each IDHi pre-treated and IDHi treatment naïve), 18 patients received ≥ 2 prior systemic treatments, and 23 patients had IDH1 mutations and 7 patients IDH2 mutations. There were no objective responses seen in the first 15 CCA patients in either cohort and enrollment was closed. The median PFS was 2.4 months 95% CI (1.9 - 6.5) and median OS 12.9 months 95% CI (6.3 – not reached). The IDHi pre-treated cohort had improved median OS compared to IDHi treatment naïve (15.3 vs 7.3, P = 0.04). There were 8/30 (27%) patients with clinical benefit (CB) as defined by PFS &amp;gt; 6 months. Patients with CB had lower baseline mean D-2HG levels compared to those without CB (1.4 umol/L vs 5.9 umol/L, p = 0.02). Whole exome sequencing revealed the study population to be enriched for HRD by sum scores and HRD mutational signatures without alternative HRD mutations other than IDH1/2. No improvement in survival was noted based on HRD status. RNA sequencing Gene Set Enrichment Analysis revealed that cell cycle and interferon alpha pathways were activated in patients with CB (p &amp;lt; 0.01). Olaparib was tolerable with the majority of adverse events grade 1-2. Conclusion: Olaparib did not result in objective responses in IDH mutated CCA. However, a subgroup of patients did demonstrate prolonged PFS comparable to historical controls for refractory CCA as well that seen with targeted agents such as IDHi. Exploratory analysis revealed this subgroup to be enriched for lower baseline 2HG levels. Serial ctDNA analysis is ongoing and will be presented. Given the CB subgroup and OS data, additional clinical trials leveraging the HRD properties of IDH mutations is warranted. Future studies may benefit from DNA damage combination therapies thereby enhancing potency. Citation Format: Michael Cecchini, Mary Jo Pilat, Nataliya Uboha, Nilofer S Azad, May Cho, Elizabeth J Davis, Jordi Rodon Ahnert, Gabriel Tinoco, Geoff I Shapiro, Simon Khagi, Benjamin Powers, Roman Groisberg, Jan Drappatz, Li Chen, Biswajit Das, Xun Bao, Jing Li, Abhijit Patel, Monica Niger, Deborah Doroshow, Diane Durecki, Scott Boerner, Ranjit Bindra, Percy Ivy, Yu Shyr, Patricia LoRusso. NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B040.

Abstract B108: NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects

Abstract Adult-type diffuse gliomas are aggressive brain cancers. The widespread standard treatment for these high-grade gliomas (HGG) is the Stupp Protocol, consisting of maximal safe surgical resection followed by concurrent chemoradiotherapy. However, since the Stupp Protocol, no significant therapeutic advances have been as widely and easily adopted, halting improvements in HGG patient survival. Therefore, identifying new drug targets or treatments that enhance the Stupp Protocol would result in therapeutic benefit. HGG are classified by distinct genetic and metabolic alterations; the most well-known being somatic gain of function mutations in isocitrate dehydrogenase (IDH) 1/2 genes. These mutations distinguish the two most severe HGGs, namely Grade 4 IDH-Mutant Astrocytoma and IDH-Wildtype Glioblastoma (GBM). IDH-mutant tumors accumulate the oncometabolite D-2-Hydroxyglutarate, which alters metabolic pathways and DNA damage responses. One metabolic alteration is decreased nicotinamide adenine dinucleotide (NAD+) levels, a metabolite essential for cellular homeostasis, and crucial in IDH-mutants as targeting NAD+ homeostasis is effective against IDH-mutant tumors. Here, we investigated if additional metabolic vulnerabilities within NAD+ and purine biosynthetic pathways could be identified in HGG cells. For this, a panel of cell lines, including an isogenic IDH1-mutant cell line, was screened for their sensitivity to a range of metabolic inhibitors in combination or not with chemo- and radiotherapy. This screen identified NUDT5, a Nudix hydrolase that metabolizes ADP-Ribose into Ribose-5-Phosphate (R-5-P) to replenish NAD+ pools, and synthesizes nuclear ATP for hormone driven chromatin remodeling, as an important determinant in glioma cell survival. Cell viability assays determined NUDT5 inhibition (NUDT5i) sensitized IDH1-wildtype HGG cells to different radiotherapy modalities, specifically X-rays and alpha particles, whilst IDH1-mutant HGG were sensitive to NUDT5i alone. Live-cell imaging analysis indicated NUDT5i induced cytostasis followed by cytotoxicity in the IDH1-mutant, suggesting IDH1-mutations confer exploitable metabolic dependencies on NUDT5 for proliferation and survival. NUDT5i did not significantly deplete NAD+ levels and NAD+ precursor supplementation failed to rescue cell survival and proliferation, indicating effects of NUDT5i are NAD+ independent. Our results indicate NUDT5 plays a crucial role in HGG proliferation and survival. Given that NUDT5 produces R-5-P for downstream nucleotide synthesis, we hypothesize that NUDT5i impairs nucleotide homeostasis and leads to replication stress. Current investigations include nucleoside supplementation and characterization of replication stress signaling by western blotting. We anticipate our results to prelude further investigations into NUDT5 as a potential therapeutic target in HGG. We suggest NUDT5i could enhance the effectiveness of the Stupp Protocol regardless of IDH-Status and advance HGG therapies. Citation Format: Thomas J.R Cox, Eren Ozcagli, Giuseppe Schettino, Lisiane B Meira. NUDT5 inhibition differentially affects IDH1-Wildtype and Mutant high-grade glioma to induce NAD+ independent radiosensitization and anti-proliferative effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B108.

Neuro-oncology Treatment Strategies for Primary Glial Tumors.

Primary brain tumors underwent reclassification in the 2021 World Health Organization update, relying on molecular findings (especially isocitrate dehydrogenase mutations and chromosomal changes in 1p, 19q, gain of chromosome 7 and loss of chromosome 10). Newer entities have also been described including histone 3 mutant midline gliomas. These updated pathologic classifications improve prognostication and reliable diagnosis, but may confuse interpretation of prior clinical trials and require reclassification of patients diagnosed in the past. For patients over seventy, multiple studies have now confirmed the utility of shorter courses of radiation, and the risk of post-operative delirium. Ongoing studies are comparing proton to photon radiation. Long term follow up of prior clinical trials have confirmed the roles and length of chemotherapy (mainly temozolomide) in different tumors, as well as the wearable novottf device. New oral isocitrate dehydrogenase inhibitors have also shown efficacy in clinical trials.

Diffusion along the perivascular space as a potential biomarker for glioma grading and isocitrate dehydrogenase 1 mutation status prediction.

The diffusion tensor image analysis along the perivascular space (DTI-ALPS) may have the potential to reflect glymphatic dysfunction in patients with glioma. The study aimed to determine the correlation of DTI-ALPS with glioma grade and isocitrate dehydrogenase 1 (IDH1) genotype and to then compare the ALPS index with other diffusion metrics. In this study, 81 patients with glioma and 31 healthy controls underwent magnetic resonance imaging (MRI) examination. The ALPS-index, fractional anisotropy (FA), mean diffusivity (MD), and mean kurtosis (MK) were calculated. Comparisons were made between the left and right hemispheres and between patients and controls. IDH1 status was compared after age adjustment. The diagnostic performance of each metric was assessed via receiver operating characteristic (ROC) analysis. In patients with glioma, the ALPS-index of the hemisphere ipsilateral to glioma was significantly lower than that of the hemisphere contralateral to glioma (1.417±0.177 vs. 1.478±0.165; P=0.002), and the bilateral ALPS-index values in patients were significantly decreased compared with those in healthy controls. The ALPS-index was significantly higher in lower-grade gliomas (LrGGs) than that in glioblastomas (GBMs) (1.495±0.151 vs. 1.320±0.159; P<0.001) and was significantly lower in IDH1-wild-type LrGGs than in IDH1-mutant LrGGs (1.400±0.185 vs. 1.530±0.123; P=0.036). FA, MD, and MK also showed significant differences between LrGGs and GBMs and between IDH1-mutant and IDH1-wild-type LrGGs (P<0.05). Furthermore, the combination of the ALPS-index with FA, MD, or MK, exhibited superior discrimination ability compared to each metric used alone. The ALPS-index combined with MD had the highest area under the curve (AUC) of 0.854 as compared to that of 0.614-0.807 for a single metric in glioma grading, while for IDH1 mutation prediction, this combination had the highest AUC of 0.861 as compared to that of 0.707-0.778 for a single metric. The reduced ALPS-index may reflect tumor-induced glymphatic system impairment, and the ALPS-index may be able to complement conventional diffusion metrics in glioma grading and IDH1 genotyping.

An Intra- and Inter-Modality Fusion Model with Invariant- and Specific-Constraints Using MR Images for Prediction of Glioma Isocitrate Dehydrogenase Mutation Status

In the 2021 World Health Organization classification of gliomas, it is proposed that Isocitrate Dehydrogenase (IDH) plays a key role. The prognosis of glioma is largely affected by IDH mutation status. Therefore, IDH mutation status needs to be predicted in advance before surgery. In the past decade, with the development of machine learning, more and more machine learning methods, especially deep learning methods, have been applied to the development of computer-aided diagnosis systems. At present, in this field, many deep learning and radiomics based methods have been proposed for IDH prediction using multimodal Magnetic Resonance Imaging (MRI). In this study, we proposed an intra- and inter-modality fusion model with invariant- and specific- constraints to improve the performance of IDH status prediction. First, MRI-based radiomics features were fused with deep learning features in each modality (intra-modality fusion) and then the features extracted from each modality of brain MRI were fused by using an inter-modality fusion model with invariant and specific constraints. We experimented our proposed method on the dataset provided by the Affiliated Hospital of Zhengzhou University in Zhengzhou, China and demonstrated the effectiveness of the proposed method. In our study, we propose two inter-modality fusion models, and our experimental results show that our best proposed method outperformed state-of-the-art methods with an accuracy of 0.79, precision of 0.80, recall of 0.75, and F1 score of 0.78. Thus, we predicted the IDH mutation status for glioma treatment with a 2% increase in accuracy and 4% increase in precision to predict the IDH mutation status for glioma treatment.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.

Is Allogeneic Hematopoietic Stem Cell Transplant (HCT) in First Remission Beneficial to Acute Myeloid Leukemia (AML) Patients with Isocitrate Dehydrogenase(IDH) Mutations?

Introduction Somatic mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) are relatively frequent in acute myeloid leukemia (AML) but with heterogeneous prognoses. The prognostic impact of this AML subtype with IDH mutations may be influenced by the comutational status with other somatic mutation, the specific types of IDH1/2 mutation (ie, IDH1 R132, IDH2 R140, and IDH2 R172). Allogeneic stem cell transplant allogeneic HCT represents a curative option for subgroups of AML patients (pts), but not for all subtypes. Transplant referral is made based on the ELN risk group classified into favorable, intermediate, and adverse risk. Although many AML pts with IDH1/2 mutations are referred to allogeneic hematopoietic stem cell transplantation (HCT) and transplanted, however it is not fully elucidated whether allogeneic HCT brings definite survival benefit to this group of the pts. Statistical attention is to be paid to the time to HCT from CR1 achievement. Before HCT, the case should be counted as a case of no HCT, while after HCT this case can be accounted as the case received HCT using the Mantel-Byar test (MBT). To handle immortal time bias, the Mantel-Byar method was used to depict outcomes during the follow-up period. Kaplan-Meier curves were avoided as they can introduce immortal bias, considering the period of time before treatment as part of the treatment arm. Instead, the MBT and landmark methods were employed to address this issue. Allogeneic HCT was treated as a time-dependent variable to prevent immortal bias. The aim of the present study is to identify if AML pts with IDH mutations obtain survival benefit from allogeneic HCT in CR1 using HCT as a time-dependent covariate with using Mantel-Byar test. Patients &amp; Method We conducted a retrospective study that included 1,228 adult pts who were diagnosed with AML and achieved CR1 in 5 centers (Toronto, Vancouver, and Montreal, Canada; Hwasun and Seoul, South Korea) from January 1997 to January 2020. Next-generation sequencing was performed in each institution according to their own standard targeted sequencing panel. We considered allogeneic HCT in CR1 as a time-dependent covariate and used a Mantel-Byar test to avoid immortal bias. Relapse-free survival (RFS) was selected for the primary endpoint, defined as the time from CR1 to relapse or death from any cause. EZR software version 1.41 was used throughout the study for statistical analysis. Results Out of 1228, a total of 238 pts (19.4%) was found to have IDH1/2 mutation, among whom 92 pts (38.6%) had isolated IDH1 mutations, 140 pts (58.8%), isolated IDH2 mutations, and 6 pts (2.5%) having coexistence of IDH1 and IDH2 mutations. With a median follow-up duration of 20.8 months (mo) (1-194.3 mo) among survivors after achieving CR1, 91 pts died while 115 pts experienced relapse. One hundred twenty-one pts received HCT in CR1, and 44 pts relapsed after HCT. The 2-year overall survival of those 238 pts was 58.9% (51.3-65.6), and 2-yr RFS was48.9% (41.6-55.8) Between the HCT (n=121) vs non-HCT group (n=117), there were no differences in sex, cytogenetic risk or other somatic mutation except NPM1 mutation which was more frequently detected in the non-HCT group (p =0.002). As expected, the HCT group was younger (55 years (15-74 yrs)) than non-HCT group (62 years (27-82 yrs), p&amp;lt;0.001). First, the RFS and OS was compared between HCT vs non-HCT groups considering HCT as non-time dependent covariate. The 2-year RFS rate was higher in the HCT group (61.0%) compared to the non-HCT group (37.0%, p= 0.0002). However, it was not translated into superior OS in the HCT group: 2-year OS rate, 64.4% in the HCT group vs 53.7% in the non-HCT group, p=0.1). We have conducted time-dependent analysis taking taken HCT in CR1 into account as a time-dependent (td) covariate using MBT. The trend of superior RFS in HCT group was reproduced in MBT: HCT in CR1 was found to improve RFS (Hazard ratio [(HR) of 0.62, (95% C.I. [0.42-0.92]; p=0.02)] as shown in Fig.1B utilizing Simon-Makuch plot. However, Multivariate analysis (MVA) for RFS did not confirm HCT in CR1 as an independent prognostic factor for RFS, while Following factors were found to be associated with RFS adversely: age &amp;gt; 60 years, presence of DNMT3A mutation, and presence of IDH1 mutation. Conclusion Allogeneic HCT in CR1 could bring some survival benefit on RFS in AML pts with IDH1/2 mutation, but not confirmed in MVA. Further study is warranted to reach a clearer conclusion with larger number of

Despite High Rates of Remission, Treatment of IDH-Mutant AML with Hypomethylating Agents and Venetoclax Is Associated with Frequent Treatment Delays, Modifications, and Transfusions

Introduction: 25-30% of adult patients with AML have mutations in the isocitrate dehydrogenase (IDH) genes, IDH1 or IDH2. Hypomethylating agents (HMA) and venetoclax (Ven) is a widely used combination in induction-ineligible patients with IDH mutations. A subset analysis of VIALE-A data showed composite complete remission or complete remission with incomplete hematologic recovery (CR/CRi) rate of 79% and median overall survival (mOS) of 24.5 months with frontline azacitidine and Ven. IDH1-mutant and IDH2-mutant patients had CR/CRi rates of 66.7% and 86.0%, respectively. IDH1-mutant patients had mOS of 15.2 months and IDH2-mutant patients did not reach mOS. We report a single-center retrospective analysis of HMA/Ven in patients with IDH mutations. Methods: This single-center retrospective study examined all patients with IDH mutations who received HMA/Ven from January 1, 2018 to June 30, 2023 at Memorial Sloan Kettering Cancer Center and summarizes their characteristics and outcomes. Demographics, adverse events, treatment course, and laboratory and transfusion data were retrospectively reviewed with institutional IRB approval. Results: Ninety-four patients, 50 with newly diagnosed (ND) AML and 44 with relapsed/refractory (R/R) AML, were identified (Table 1). 32% (n=16) of ND patients and 41% (n=18) of R/R patients had IDH1 mutations and 68% (n=34) of ND patients and 59% (n=26) of R/R patients had IDH2 mutations. With median follow-up of 21.9 months, mOS was 29.2 months (95% CI 17.9, NA) for ND patients and 17.1 months (95% CI 12.1, 34.2) for R/R patients (Figure 1). The CR/CRi rate was 76% (n=38; 30% CR, n=15; 46% CRi, n=23) for ND patients and 52% (n=23; 32% CR, n=14; 20% CRi, n=9) for R/R patients. Patients received median of 3 cycles of HMA/Ven (range 1, 22) and experienced frequent treatment modifications and delays. Of 356 total cycles, 62% (n=218) were modified to have fewer than 28 days of venetoclax, 7 days of azacitidine, or 5 days of decitabine. In addition, 296 cycle holds or delays of at least one week occurred. 66.2% (n=196) of holds or delays were due to cytopenia and 11.5% (n=34) were due to infection. 77% (n=72) of patients had Common Terminology Criteria for Adverse Events (CTCAE) grade 3 pancytopenia and 47% (n=44) had febrile neutropenia. Through 356 cycles spanning 21,386 patient-days, 5,651 complete blood counts (CBCs, mean 15.9 per cycle), 986 red blood cell transfusions, (RBC, mean 2.8 per cycle) and 1,018 platelet transfusions (PLT, mean 2.9 per cycle) were recorded at our center. Additional CBCs or transfusions performed externally were not captured. After CR/CRi, dose adjustments, cycle holds or delays, and cytopenias remained frequent. The 63 patients that achieved CR/CRi underwent 198 cycles, 71% (n=141) of which were modified as above. There were 209 holds or delays (65%, n=136 for cytopenias, 10%, n=21 for infection). Median time between cycles was 7 weeks. Cycles given after CR/CRi frequently resulted in grade 3 cytopenias by CTCAE (neutropenia: 82%, n=163; anemia: 15%, n=29; thrombocytopenia: 37%, n=74). Through 198 cycles spanning 13,463 patient-days, 2,426 CBCs (mean 12.3 per cycle), 141 RBC transfusions, (mean 0.7 per cycle) and 236 PLT transfusions (mean 1.2 per cycle) were performed. At last follow-up, 28% (n=14) of ND AML patients continued HMA/Ven, whereas 28% (n=14) had proceeded to allogeneic stem cell transplantation (alloSCT), 28% (n=14) were relapsed or refractory to HMA/Ven, and 12% (n=6) were deceased. For R/R AML patients, 20% (n=9) continued HMA/Ven but 27% (n=12) had proceeded to alloSCT, 34% (n=15) were relapsed or refractory, and 14% (n=6) were deceased. Of the 68 patients who were no longer receiving HMA/Ven at end of follow-up, only 23.5% (n=16) of patients received an IDH-inhibitor in their next line of therapy, the rest primarily transitioning to alloSCT (38.2%, n=26), chemotherapy (8.8%, n=6), or supportive measures (20.6%, n=14). Conclusion: In a single-center retrospective setting, HMA/Ven is efficacious in achieving CR/CRi in ND and R/R IDH-mutant AML. However, its use, even in CR/CRi, is associated with significant cytopenias and infections that necessitate indefinite and frequent treatment modification, laboratory utilization, and transfusions. A strategy that incorporates HMA/Ven followed by non-myelosuppressive targeted therapy with IDH inhibition as maintenance should be explored to minimize these toxicities.

Identification of 17 Distinct IDH1/IDH2 Mutations from Acute Myeloid Leukemia Peripheral Blood Using a Multiplex Real-Time PCR Closed Cartridge System

Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated “blast cells” in peripheral blood and bone marrow causing ineffective erythropoiesis and bone marrow failure. It has been demonstrated that mutations in isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) are associated with disease relapse and reduced survival. There are 17 AML-related IDH1/IDH2 mutations located at R100, R132, R140, and R172 that are targeted by different IDH1/IDH2 selective inhibitors. The sensitivity of detecting IDH1/IDH2 needs to be improved for early cancer detection. Current PCR-based assays cannot detect all 17 IDH1/IDH2 mutations, and next generation sequencing approaches take longer, use complicated workflows, and require costly reagents as well as substantial data analysis. Therefore, the rapid identification of patients with AML who harbor IDH1/IDH2 mutations could improve the efficient selection of targeted inhibitors for these patients. Aim: To create a multiplex PCR assay that detects 17 IDH1/IDH2 mutations from peripheral blood with high sensitivity and specificity. Method: We will highlight five technical aspects of this qPCR assay that contribute to its high sensitivity and specificity: 1. the “anchor - bridge - foot” design of SuperSelective (SS) primers that can detect different mutations within the same codon. 2. the amplification-refractory mutation system (ARMS) primer design strategy was also tested which has comparable effects to SS primer. 3. a two-step nested PCR to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 4. a short (20-25nt) blocking oligonucleotide (wild-type blocker, WTB) to reduce SS and ARMS primer binding at the wild-type codon site. 5. four probes carrying different fluorescence reporters were used to identify the four IDH1/IDH2 mutation codons. Results: We synthesized 17 plasmid DNA (pDNA) templates and 14 CRISPR-engineered cell lines, each carrying one IDH1 or IDH2 mutation and the housekeeping gene ABL. SS and ARMS primer were screened with 5·10 4 cps/test of mutation template in a whole blood background. 6 mutations of R132 Codon (R132C, G, H, L, S, V), R100Q, 4 mutations of R140 (R140Q, L, G, W), and 6 mutations of R172 (R172K, M, G, S1 [516G&amp;gt;T], S2 [516G&amp;gt;C], W) can successfully be identified from whole blood lysate at the LoD level (1%) as shown in Figure 1 in red. Moreover, adding WTB to the PCR reaction improved the specificity with a reduced background signal. Importantly, in addition to the plasmid, cell line genomic DNA (gDNA) that was spiked into blood lysate at the LoD level (1%) also performed similarly to plasmid DNA as shown in Figure 1 in blue. Summary/Conclusion: We present a prototype assay with design intention to detect 17 mutations of IDH1/IDH2 from the peripheral blood of AML patients with excellent sensitivity and specificity and a strong potential to outperform any products on the market. Utilizing our Xpert Instrument System a fully automated, single-use cartridge product with pre-loaded sample-processing reagents, the assay aims to report results in less than 3 hours, including hands-on time.

Circulating Oncometabolite 2-hydroxyglutarate as a Potential Biomarker for Isocitrate Dehydrogenase (IDH1/2) Mutant Cholangiocarcinoma.

Isocitrate dehydrogenase (IDH) enzymes catalyze the decarboxylation of isocitrate to alpha-ketoglutarate (αKG). IDH1/2 mutations preferentially convert αKG to R-2-hydroxyglutarate (R2HG), resulting in R2HG accumulation in tumor tissues. We investigated circulating 2-hydroxyglutate (2HG) as potential biomarkers for patients with IDH-mutant (IDHmt) cholangiocarcinoma (CCA). R2HG and S-2-hydroxyglutarate (S2HG) levels in blood and tumor tissues were analyzed in a discovery cohort of patients with IDHmt glioma and CCA. Results were validated in cohorts of patients with CCA and clear-cell renal cell carcinoma. The R2HG/S2HG ratio (rRS) was significantly elevated in tumor tissues, but not in blood for patients with IDHmt glioma, while circulating rRS was elevated in patients with IDHmt CCA. There were overlap distributions of circulating R2HG and total 2HG in patients with both IDHmt and wild-type (IDHwt) CCA, while there was minimal overlap in rRS values between patients with IDHmt and IDHwt CCA. Using the rRS cut-off value of 1.5, the sensitivity of rRS was 90% and specificity was 96.8%. Circulating rRS is significantly increased in patients with IDHmt CCA compare with patients with IDHwt CCA. Circulating rRS is a sensitive and specific surrogate biomarker for IDH1/2 mutations in CCA. It can potentially be used as a tool for monitoring IDH-targeted therapy.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Introduction: The efficacy of chimeric antigen receptor (CAR) T-cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Mitochondria are vital for the regulation of memory T-cell formation or exhaustion. A mitochondria-related compound screening was performed and we found that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances long-lived memory CAR T-cell formation, and improves tumor clearance in vivo. IDH2 as the key component of the tricarboxylic acid (TCA) cycle, we hypothesized that IDH2 inhibition might promote CAR T-cell persistence by reprogramming the metabolism. The study aims to systematically evaluate the role and the mechanism of IDH2 inhibition on CAR T cells, and explore the application of enasidenib in CAR T-cell therapy. Methods and Results: To identify mitochondrial components that affect the long-term efficacy of CAR T-cells, we performed an in vitro screening using a mitochondria-related compound library based on the enrichment of the CD62L + CAR T-cell subset, which mainly contains T memory stem cells (T SCM) and central memory T cells (T CM). Among several candidate compounds that promoted memory CAR T-cell formation, enasidenib (ENA), an inhibitor of both wild-type and mutant IDH2 enzymes, was the most effective one. The proportion of CD62L has increased by over 20%. To determine the effect of ENA on exhaustion induced by tonic CAR signaling and tumor antigen stimulation, we measured the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 and the level of apoptosis in freshly expanded CAR T cells and B-ALL encountered CAR T cells. CAR T cells treated with ENA exhibited reduced surface levels of those inhibitory receptors. We measured the production of granzyme B and IFNγ, and tumor rechallenge assay in CAR T cells after ENA treatment to assess the effector function, ENA treated CAR T cells exerted enhanced and sustained cytotoxicity in a tumor rechallenge assay at an extreme E:T ratio of 1:10. These results were observed not only in CD19-41BBζ CAR T cells, but also in CD19-28ζ and GD2-28ζ CAR T cells. CAR T cells expanded in the presence of ENA significantly prolonged survival of recipients after Nalm-6 infusion (p &amp;lt; 0.0001), extended in vivo ENA treatment further enhanced CAR T-cell expansion and tumor suppression. The median survival was extended from 45 to 67.5 days. Consistent with the effect of ENA treatment, IDH2 knockdown increased the proportions of the T scm/T n and T cm subsets, and alleviated CAR T-cell exhaustion. IDH2-knockdown CAR T cells had sustained cytotoxicity and higher CD62L expression after rounds of killing B-ALL cells in vitro. To systematically explore the effects of IDH2 inhibition on the metabolism of CAR T cells, relative metabolite amounts was measured using mass spectrometry (MS). IDH2-inhibited CAR T cells exhibited a substantial reduction of metabolites in the TCA cycle, including succinate, fumarate, and malate, and in the glycolysis pathway, including lactate, phosphoenolpyruvate. Mechanistically, IDH2 inhibition reprogram central carbon metabolism of CAR T cells by redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway. In addition, IDH2 limits cytosolic acetyl-CoA level to prevent histone acetylation that promotes memory cell formation. Significance: Our study indicates that metabolic intervention in CAR T cells with the FDA-approved IDH2 inhibitor enasidenib can advance current treatment, with better tumor eradication and CAR T-cell persistence.

Molecular Measurable Residual Disease in Patients with Newly Diagnosed m IDH1 Acute Myeloid Leukemia Treated with Ivosidenib + Azacitidine

Background: Ivosidenib (IVO) + azacitidine (AZA) improved complete remission (CR) rates and overall survival (OS) relative to placebo (PBO) + AZA in patients (pts) with newly diagnosed mutant isocitrate dehydrogenase 1 (m IDH1) acute myeloid leukemia (AML) in the pivotal AGILE study (Montesinos et al. NEJM 2022). Prior analyses showed clinical responses to IVO+AZA were associated with deep clearance of m IDH1 (limit of detection [LOD]: 0.02-0.04% variant allele frequency [VAF]), as well as clearance of baseline co-mutations below the threshold of conventional next-generation sequencing (NGS) (LOD: 2.0% VAF) (Döhner et al. Blood 2022; Daigle et al. Blood 2021). Measurable residual disease (MRD), a negative prognostic marker, following chemotherapy is often detected via multiparametric flow cytometry or quantitative polymerase chain reaction although both techniques have their limitations. (Heuser et al. Blood 2021). NGS can detect a wide range of AML mutations and studies show that NGS-assessed MRD is highly predictive of both relapse and survival (Thol et al. Blood 2018; Tsai et al. Blood Adv 2021). However, a limitation of NGS is difficulty in distinguishing between somatic leukemia-associated mutations and clonal hematopoiesis of indeterminate potential (CHIP) (Bacher et al. Blood Cancer J 2018). We aimed to perform an NGS analysis of MRD responses in the AGILE study. Methods: All AGILE pts who had a best overall response (BOR) of CR, CR with incomplete count recovery, or CR with incomplete platelet recovery, and had ≥1 on-treatment bone marrow mononuclear cell (BMMC) sample available were included. Suggested timepoints for MRD testing included day 1 of treatment cycles 3, 5, 7, 9, 11, 14, 20, 26, and 32. Baseline and on-treatment BMMC DNA samples were analyzed using a diagnostic 51-gene myeloid NGS panel (LOD: 3% VAF) and a 26-gene AML MRD panel, respectively. For variants detected at baseline, the LOD was 0.1% VAF; for variants not detected at baseline (either emerging mutations or if baseline samples were unavailable), the LOD was 0.5% VAF. All variants above the applicable LOD with known or potential clinical significance were considered evidence of positive MRD, aside from DNMT3A, TET2 and ASXL1 (“DTA”) mutations to reduce the risk of false-positive MRD due to CHIP. Results: The analysis set comprised 173 samples from 33 IVO+AZA-treated pts and 10 PBO+AZA-treated pts (Fig. 1). Clinical response data were based on a March 18, 2021 primary analysis data cut; OS data were based on an updated June 30, 2022 data cut. The median number of baseline mutations was 4 (range: 1-10) per patient, median number of MRD assessments was 3 (range: 1-9) and median follow-up period was 189 days (range: 49-875). Ten (30.3%) IVO+AZA-treated pts became MRD-negative (MRD neg), compared with 2 (20.0%) PBO+AZA-treated pts. All pts attaining an MRD neg response had a BOR of CR (10 [34.5%] of 29 IVO+AZA-treated pts in CR; 2 [22.2%] of 9 PBO+AZA-treated pts in CR). Seven (70.0%) of the 10 MRD neg IVO+AZA-treated responding pts converted to an MRD neg response by day 1 of cycle 7 (C7D1). Fewer pts with an MRD neg response had an Adverse risk at baseline according to European LeukemiaNet 2022 genetic risk classification versus those pts with an MRD-positive (MRD pos) response (26.3% vs 73.7%) (Table 1). In the IVO+AZA arm, rates of event-free survival at 12 months and OS at 24 months were numerically higher in MRD neg vs MRD pos responders (80.0% vs 60.6% and 88.9% vs 72.3%, respectively) but did not reach statistical significance. All 5 (15.2%) IVO+AZA-treated pts with confirmed relapses had detectable MRD at the last completed assessment prior to relapse. One pt converted from an MRD neg to an MRD pos response (“MRD relapse”) 169 days prior to overt clinical relapse. Among the 23 IVO+AZA-treated pts without an MRD neg response, 16 were alive as of the updated data cut; OS duration ranged from 20.3-48.9 months. Conclusions: IVO+AZA induced molecular MRD negativity in approximately one third of responding pts with newly diagnosed m IDH1 AML; MRD neg responses were most often observed on or before C7D1. Interestingly, several pts had durable clinical responses and OS despite ongoing MRD positivity, potentially due to persistent CHIP rather than true MRD, illustrating the complexity of interpreting NGS MRD data. Additional analyses, including associations between specific molecular alterations, MRD response, and OS are ongoing and will be presented.

Olutasidenib Alone or in Combination with Azacitidine Induces Durable Complete Remissions in Patients with mIDH1 Myelodysplastic Syndromes/Neoplasms (MDS)

Introduction Olutasidenib is a potent, selective, oral, small-molecule inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1). Olutasidenib is approved in the US for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML) based on the pivotal cohort (n=153) of a registrational Phase 1/2 trial (NCT02719574), which demonstrated a rate of complete remission (CR) or CR with partial hematologic recovery (CRh) of 35%, with a duration of response of 25.9 months. The Phase 1/2 study also enrolled patients with mIDH1 myelodysplastic syndromes/neoplasms (MDS), and here we report results from this subset of 22 patients. Methods The Phase 1/2 open-label study enrolled 332 adult patients with mIDH1 AML or intermediate-, high-, or very high-risk MDSfrom 57 centers in 9 countries. Inclusion criteria included an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 and adequate liver and renal function. Patients were R/R to prior therapy or, if treatment-naïve (TN), had to be eligible for azacitidine therapy (to be enrolled in a combination therapy cohort). Olutasidenib was administered at 150 mg BID (n=3) or 100-150 QD (n=3) as monotherapy or in combination with azacitidine (n=16), given at 75 mg/mm 2 daily for 7 consecutive days, in 28-day cycles. The primary efficacy endpoint for MDS was the CR rate, based on response criteria of the International Working Group in MDS. Secondary efficacy endpoints included overall response (CR/PR/marrow CR), time to response, duration of response and safety. Results Twenty-two patients with MDS were enrolled, including 6 who received monotherapy (4 R/R and 2 TN) and 16 who received combination therapy (11 R/R and 5 TN). Baseline demographics and disease characteristics are shown in Table 1. The age ranged from 59-87 years, and 59% of the study population were male. Seven patients were treatment-naïve and 15 were R/R with 1-4 prior treatment regimens. For the pooled Phase 1 and 2 data (n=22), 6 (27%) patients achieved CR and 7 (32%) patients achieved marrow CR with no PRs, generating a 59% overall response rate. The median time to response was 2.0 months. The median duration of response was not reached (NR) at 30.1+ months ( Table 2). The overall response rate was 2/6 (33%) for monotherapy and 11/16 (69%) for combination therapy. Three of 6 patients received monotherapy at a lower dose (100-150 QD) than the approved dose level, and none responded. Of the 3 patients who received monotherapy at the approved dose level (150mg BID), 2 (66%) responded. In the 7 TN patients, the overall response rate was 86%. In the 15 R/R patients, the overall response rate was 47%. All patients with MDS experienced at least 1 treatment-emergent adverse event (TEAE). The most frequent TEAEs in the study were nausea, constipation, vomiting, thrombocytopenia, neutropenia, diarrhea, and fatigue. Grade 3 TEAEs occurred in 19/22 (86%) patients, and Grade 4 TEAEs in 9/22 (41%). The most frequent Grade 3/4 TEAEs reported were cytopenias. Grade 3 transaminitis occurred in 4 patients. Grade 3 differentiation syndrome was reported in 1 patient, who discontinued treatment after 23 days due to pneumonia, which lead to respiratory failure and death. TEAEs resulting in death occurred in 5 patients and included disease progression (3), pneumonia (1, mentioned above) and chest fungal infection (1). The overall safety profile in patients with MDS was consistent with what has been reported in patients with AML in this study (Watts, et al., Lancet Hematol, 2022; de Botton et al., Blood Adv, 2023). Conclusions Olutasidenib, both as monotherapy and in combination with azacitidine, induced durable remissions in patients with intermediate-, high-, or very high-risk MDS. Patients had varying treatment backgrounds, including treatment-naïve and up to four prior regimens. This treatment had a tolerable and manageable safety profile. These encouraging results, which warrant further investigation with a larger number of patients, show that olutasidenib has clinically meaningful activity in patients with mIDH1 MDS.

Immunosuppression in Isocitrate Dehydrogenase Mutated Acute Myeloid Leukemia

Introduction: Mutations in the isocitrate dehydrogenase (IDHmut) enzymes 1 and 2 are prevalent in a diverse array of human malignancies, including melanoma, glioma, acute immunoblastic T cell lymphoma (AITL) and acute myeloid leukemia (AML,15-20% of patients). IDHmut enzymes produce R-2-hydroxyglutarate (R-2-HG), an oncometabolite with immunomodulatory capability (Notarangelo et al, Science 2022). Immunotherapies in AML outside of allogeneic stem cell transplantation have proven to be largely ineffective, possibly due to immune evasion mechanisms intrinsic to myeloid malignancies. Herein, we aim to characterize the immune repertoire in IDHmut AML patients at the time of diagnosis, as well as the mechanisms by which R-2-HG may suppress immune cell function. Methods: The immune repertoire in cryopreserved peripheral blood mononuclear cells (PBMCs, n=10) and bone marrow mononuclear cells (BMMCs, n=8) of IDHmut AML patients were characterized using full spectrum flow cytometry to evaluate 30 different immune cell markers. T cell activation (CD25, CD69, CD40L, CD134), exhaustion (PD-1, CTLA-4, Lag-3, Tim-3), differentiation (Foxp3, CXCR3, CCR4, CCR6), and memory (CD45RA, CCR7, CD28, CD95) markers were assessed. IDH wild-type (IDHwt) AML PBMCs (n=4), BMMCs (n=12) and healthy volunteer PBMCs (n=6) and BMMCs (n=3) were used as controls. To investigate the effects of R-2-HG on CD4 + T cell activation, primary human naïve CD4 + T cells (CD4 +, CD45RA +) were isolated from healthy donors and activated (anti-CD3/CD28) in the presence of R-2-HG (20 mM) or PBS control. Activation, proliferation and cytokine production were assessed with flow cytometry, intracellular cytokine staining (ICS) and LEGENDplex at 4, 8, 24 and 72 hrs. The OT-II transgenic mouse model was used to assess antigen-specific T cell activation. T cell metabolism at rest and after activation in the presence of R-2-HG was evaluated using SCENITH (Argüello et al, Cell Metab 2021) and 13C 6-glucose isotope labeling coupled with mass spectrometry (MS) performed by the UCLA Metabolomics Center. Results: Full-spectrum flow cytometry analysis revealed a trend for higher CD4:CD8T cell ratio in AML BMMCs (fold=1.95, p=0.229) as well as increased CD4 + T cell activation and exhaustion markers compared to healthy donor BMMCs. No appreciable differences in CD4 + T cell markers were found between IDHwt AML and IDHmut AML, likely due to AML heterogeneity and small sample size. Nevertheless, our preliminary results suggest CD4 + T cell engagement in AML; thus, we chose to investigate how CD4 + T cells are affected by R-2-HG. Primary human naïve CD4 + T cells activated in the presence of R-2-HG exhibited a defect in early activation demonstrated by decreased CD69 expression (n=7, fold=0.773, p=0.001) and concomitant decreased production of IL-2 measured by ICS (n=7, fold=0.403, p=0.0001) and LEGENDplex (n=3, fold=0.557, p=0.046). Proliferation in the presence of R-2-HG was inhibited in a dose dependent manner (n=6, Division Index fold change=0.768, p=0.006 at 20mM). Antigen-specific OT-II CD4 + T cells showed a decrease in proliferation in response to OVA 323-339 (n=3, Division Index fold change=0.620, p=0.004). Given R-2-HG's structural similarity to α-ketoglutarate (αKG), we hypothesized that disruption in CD4 + T cell activation when cultured with R-2-HG may be due to metabolic dysfunction. Indeed, we found a decrease in global metabolism in CD4 + T cells cultured with R-2-HG (fold = 0.678, p = 0.009). MS revealed decreased levels of succinate and malate downstream and accumulation of αKG upstream of α-ketoglutarate dehydrogenase (KGDH) in the tricyclic acid (TCA) cycle, suggesting a defect in TCA flux (fold succinate / αKG ratio =0.134 unactivated, p = 0.029; 0.231 4h activation, p = 0.012; 0.328 24h activation, p = 0.004; Figure 1). Moreover, MS also showed increased amounts of essential amino acids (Ile, Leu, Met, Phe, Thr, Trp, Val, His; Figure 2), suggesting a compensatory reliance on amino acid import and metabolism. Conclusions: By characterizing primary AML samples at diagnosis, we found trending increases in CD4:CD8 ratio and expression of CD4 + T cell activation and exhaustion markers compared to healthy controls. We also demonstrate R-2-HG suppression of CD4 + T cells, potentially via inhibition of KGDH in the TCA cycle. Our studies show R-2-HG in IDHmut tumors can modulate CD4 + T cell function and contribute to immune escape in human AML.

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.