Mutant Isocitrate Dehydrogenase Enzymes Research Articles

Vorasidenib- A Paradigm Shift in IDH1- or IDH2-Mutant Low-Grade Glioma Treatment.

Low-grade gliomas (LGGs) pose significant challenges in cancer research and treatment due to their infiltrative nature and tendency for recurrence, making them the most common brain tumour in adults. Most of the LGGs have the propensity to cause notable alterations in the microenvironment of the tumour thus making them difficult to treat. These alterations can be attributed to the prevalent mutations in the genes that produce isocitrate dehydrogenase (IDH) enzymes, especially in grade 2 diffuse gliomas. [1] Despite its inability to provide a complete cure, chemoradiation has been adopted as the standard therapy in grade 3 gliomas since it can lead to long-lasting remission. Chemoradiotherapy, when administered promptly, can help prevent tumour progression, and recurrence after surgery and improve long-term outcomes. Nevertheless, immediate adjuvant chemoradiotherapy is not administered in patients afflicted with tumours containing IDH mutant grade 2 gliomas. This decision is often made to circumvent potential harm such as radiation-induced cognitive problems, chemotherapy-related DNA mutations, and other negative effects on their health. [2] Due to the intricacies associated with the treatment decisions and the inclination to delay aggressive therapies to avoid adverse events, creates a necessity for a novel therapeutic strategy that is both effective and well tolerated. Vorasidenib, an oral medication specifically designed to deal with IDH mutations has displayed promise despite existing challenges. By aiming to specifically target aberrant metabolic pathways triggered by mutant IDH enzymes, Vorasidenib offers a potential treatment capable of interrupting tumour growth and could subsequently result in better patient outcomes. Among the many reasons that make Vorasadenib an appealing option are its capability to penetrate the blood-brain barrier and its promising safety profile, resulting in effective treatment while alleviating potential side effects. [3] Vorasidenib’s efficacy is derived from its ability to halt the metabolic cascades driven by mutant IDH enzymes thus leading to decreased progression of the gliomas. Clinical studies elicit the substantial value Vorasadenib has to offer by enhancing progression-free survival and postponing the advancement of the illness in patients with grade 2 IDH-mutant gliomas. This offers an optimistic prospect for delaying the need for more aggressive treatments in these patients. [2,4] Hence, to conclude Vorasadenib can transform treatment norms and enhance outcomes for those suffering from IDH-mutant LGG. Furthermore, it is crucial to investigate different treatment combinations, finding markers that would help assess clinical response to treatment, which in turn would be an important milestone in the treatment journey.

Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells.

Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication invitro and invivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.

Rational Computational Approaches in Drug Discovery: Potential Inhibitors for Allosteric Regulation of Mutant Isocitrate Dehydrogenase-1 Enzyme in Cancers

Mutations in homodimeric isocitrate dehydrogenase (IDH) enzymes at specific arginine residues result in the abnormal activity to overproduce D-2 hydroxyglutarate (D-2HG), which is often projected as solid oncometabolite in cancers and other disorders. As a result, depicting the potential inhibitor for D-2HG formation in mutant IDH enzymes is a challenging task in cancer research. The mutation in the cytosolic IDH1 enzyme at R132H, especially, may be associated with higher frequency of all types of cancers. So, the present work specifically focuses on the design and screening of allosteric site binders to the cytosolic mutant IDH1 enzyme. The 62 reported drug molecules were screened along with biological activity to identify the small molecular inhibitors using computer-aided drug design strategies. The designed molecules proposed in this work show better binding affinity, biological activity, bioavailability, and potency toward the inhibition of D-2HG formation compare to the reported drugs in the in silico approach.

Targeting Cell Adhesion Molecules in IDH Mutant Myeloid Malignances

Neomorphic mutations in the genes encoding isocitrate dehydrogenase (IDH) are common in human cancers including myeloid malignancies. The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by mutant IDH promotes leukemia development by competitively inhibiting α-ketoglutarate (α-KG)-dependent dioxygenases, causing epigenetic dysregulation of histone and DNA methylation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA-approved allosteric inhibitors (ivosidenib and enasidenib targeting mutant IDH1 and IDH2 respectively) are used for treating newly diagnosed and/or relapsed/refractory AML patients with IDH mutations. However, the low response rate and primary resistance to IDHmut inhibitors are major clinical challenges. Moreover, acquired resistance to IDH inhibition can occur through multiple mechanisms, including R-2-HG-restoring second-site IDH mutations and the selection of sub-clonal mutations in RUNX1/CEBPA or RAS-RTK pathway. These findings underscore the importance of developing new and more effective approaches for the treatment of IDH-mutant MDS and AML patients. To identify mutant IDH-mediated alterations and actionable dependencies, we generated isogenic TF-1 myeloid leukemia cell lines containing endogenous, hotspot IDH mutations (IDH1R132H, IDH2R140Q, IDH2R140W, and IDH2R172K) by CRISPR/Cas9-mediated base-editing. By comparative transcriptomic (RNA-seq) and epigenomic (ATAC-seq and ChIP-seq) profiling of base-edited versus parental isogenic leukemia cells, we identified cell adhesion molecules, especially CD44, as the top upregulated genes in IDH-mutant leukemia cells. CD44 expression is significantly upregulated in IDH-mutant AML patient samples relative to normal CD34+ hematopoietic stem/progenitor cells (HSPCs) or other AML samples without IDH mutations. Increased CD44 expression is also associated with overall poorer survival and therapy resistance in independent AML cohorts. Importantly, we found that ectopic CD44 expression promotes cytokine-independent cell growth of TF-1 myeloid leukemia cells, resembling the effect observed upon expression of mutant IDH. Moreover, CD44 depletion led to decreased R-2-HG levels in base-edited IDH-mutant TF-1 leukemia cells, and impaired the propagation of IDH-mutant leukemia cells in vitro and in xenotransplanted recipient mice. These studies establish direct evidence that IDH mutations cause aberrant activation of cell adhesion molecules such as CD44, and CD44 is required for the propagation of IDH-mutant myeloid leukemia cells. Mechanistically, we found that the aberrantly activated CD44 is first cleaved by matrix metalloproteinases MMP2 and MMP9 for proteolytic release of its ectodomain from membrane, followed by γ-secretase-mediated intramembranous cleavage to produce the CD44 intracellular domain (CD44ICD). CD44ICD subsequently activates the pentose phosphate pathway (PPP) for the production of NADPH, a critical cofactor for mutant IDH enzymes to convert α-KG to R-2-HG. Hence, mutant IDH-mediated activation of CD44 functions to rewire intracellular metabolism to facilitate IDHmut-catalyzed R-2-HG production, supporting a model for mutant IDH-induced feed-forward oncogenic programs. CD44 also activates AKT/ERK signaling pathway to promote the proliferation and cytokine-independent growth of IDH-mutant leukemia cells. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combination regimens consisting of IDHmut inhibitors and humanized monoclonal antibody specific for CD44 (RG7536) or MMP2/9 inhibitors (SB-3CT) invitro and invivo. Taken together, our findings uncover the cell adhesion molecule-mediated metabolic signaling as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies.

Ivosidenib in patients with IDH1-mutant relapsed/refractory myelodysplastic syndrome (R/R MDS): Updated enrollment and results of a phase 1 dose-escalation and expansion substudy.

7053 Background: Mutations in isocitrate dehydrogenase 1 ( IDH1) occur in ̃3% of patients (pts) with MDS and are associated with increased transformation to acute myeloid leukemia (AML). Ivosidenib (IVO), an oral, potent, targeted inhibitor of the mutant IDH1 (mIDH1) enzyme, is FDA approved for m IDH1 R/R AML and m IDH1 newly diagnosed AML in pts ≥75 years old or with comorbidities precluding the use of intensive induction chemotherapy. In the first-in-human study of IVO in pts with m IDH1 advanced hematologic malignancies (NCT02074839), 12 pts with R/R MDS received IVO 500 mg once daily (QD). Based on encouraging safety and efficacy findings, including an investigator-assessed overall response rate (ORR) of 75%, with median response duration of 21.4 months, the FDA granted Breakthrough Therapy designation to IVO in m IDH1 R/R MDS and the study was amended to enroll additional pts. We report updated results. Methods: This substudy of the single-arm, open-label study of IVO evaluated pts with R/R MDS after documented failure or relapse following prior standard therapy including intensive chemotherapy and hypomethylating agents. Other key eligibility criteria included: high disease burden based on IPSS or IPSS-R risk at baseline; an Eastern Cooperative Oncology Group performance status score of 0–2; and no prior IDH1 inhibitor therapy. Pts received IVO 500 mg QD orally on days 1–28 of 28-day cycles. Results: As of 08May2021, 16 pts with R/R MDS were enrolled: 5 (31%) pts remained on treatment and free from leukemic transformation; 11 (69%) had discontinued including 6 for disease progression, 1 for allogeneic stem cell transplantation, and 1 owing to an adverse event (AE) of sepsis (the only fatal AE; reported by investigator as not related to IVO). AEs are summarized in the Table. 2 pts each experienced differentiation syndrome (grade 2) and QTcF prolongation (grade 1 and 2). 7/16 pts achieved complete response (CR, 44%; 95% CI, 20%, 70%), 1 achieved partial response (6%), and 5 achieved marrow CR (31%), resulting in an ORR of 81% (95% CI, 54%, 96%). Hematologic improvement in ≥1 lineages was achieved by 11/16 (69%) pts. The Kaplan-Meier estimate of duration of CR+PR at 12 months was 60%. 3 pts experienced CRs lasting 24.0, 63.7, and 65.4 months, which remain ongoing. 5/7 pts (71%) who were transfusion dependent at baseline became independent of red blood cell or platelet transfusions for 56 or more consecutive days on treatment. Additional translational data are being analyzed. Conclusions: In pts with m IDH1 R/R MDS, IVO monotherapy was tolerable and induced durable remissions and transfusion independence. These findings support the role of IVO as an effective, oral, targeted treatment for pts with m IDH1 R/R MDS. Clinical trial information: NCT02074839. [Table: see text]

AGILE: A Global, Randomized, Double-Blind, Phase 3 Study of Ivosidenib + Azacitidine Versus Placebo + Azacitidine in Patients with Newly Diagnosed Acute Myeloid Leukemia with an IDH1 Mutation

AGILE: A Global, Randomized, Double-Blind, Phase 3 Study of Ivosidenib + Azacitidine Versus Placebo + Azacitidine in Patients with Newly Diagnosed Acute Myeloid Leukemia with an IDH1 Mutation

MDS-335: Ivosidenib (IVO) in Patients with IDH1-Mutant Relapsed/Refractory Myelodysplastic Syndrome (R/R MDS): Updated Enrollment for the MDS Sub-Study

Context: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in ~3% of patients with MDS and are associated with increased transformation to acute myeloid leukemia (AML). IVO is an oral, potent, targeted inhibitor of the mutant IDH1 (mIDH1) enzyme and is FDA-approved for mIDH1 R/R AML and mIDH1 newly diagnosed AML in patients ≥75 years old or with comorbidities precluding the use of intensive induction chemotherapy. In the first-in-human, phase 1 study of IVO in patients with mIDH1 advanced hematologic malignancies (NCT02074839), 12 patients with R/R MDS, with median age 72.5 years (range 52–78), received IVO 500 mg once daily (QD). All patients received prior MDS treatment. Investigator-assessed ORR (CR + PR + marrow CR, per IWG 2006) was 75% (95% CI 43–95), with median duration of response of 21.4 months (95% CI 2.3–NE). Nine (75%) patients were transfusion-independent for ≥56 days during treatment. No dose-limiting toxicities or adverse events leading to treatment discontinuation were reported among patients with MDS. Based on these encouraging data, the FDA granted Breakthrough Therapy Designation to IVO in mIDH1 MDS; the study was amended to enroll additional patients with mIDH1 R/R MDS. Objective: To evaluate safety, tolerability, clinical activity, and pharmacokinetics/pharmacodynamics of IVO in patients with mIDH1 R/R MDS. Design: A sub-study of the single-arm, open-label, phase 1 dose escalation and expansion study of IVO in mIDH1 advanced hematologic malignancies, evaluating patients with R/R MDS. Patients must have R/R disease after prior standard therapy; high disease burden based on cytopenia and/or transfusion dependence at baseline; an Eastern Cooperative Oncology Group performance status score of 0–2; and be amenable to bone marrow aspirate and/or core biopsy at specified study timepoints. Patients with documented AML are not eligible. IVO 500 mg QD orally on days 1–28 of 28-day cycles. Results: Study is open; enrollment of ~23 patients from the US and France planned. Results not yet available. Conclusions: This sub-study will provide additional insights into safety, tolerability, clinical activity, and pharmacokinetics/pharmacodynamics of treatment with IVO in patients with mIDH1 R/R MDS. Funding: Agios; Servier.

Dual-Knockout of Mutant Isocitrate Dehydrogenase 1 and 2 Subtypes Towards Glioma Therapy: Structural Mechanistic Insights on the Role of Vorasidenib.

Recently, Vorasidenib (AG-881) has been reported as a therapeutic alternative that exerts potent dual inhibitory activity against mIDH1/2 towards the treatment of low-grade glioma. However, structural and dynamic events associated with its dual inhibition mechanism remain unclear. As such, we employ integrative computer-assisted atomistic techniques to provide thorough structural and dynamic insights. Our analysis proved that the dual-targeting ability of AG-881 is mediated by Val255/Val294 within the binding pockets of both mIDH1 and mIDH2 which are shown to elicit a strong intermolecular interaction, thus favoring binding affinity. The structural orientations of AG-881 within the respective hydrophobic pockets allowed favorable interactions with binding site residues which accounted for its high binding free energy of -28.69 kcal/mol and -19.89 kcal/mol towards mIDH1 and mIDH2, respectively. Interestingly, upon binding, AG-881 was found to trigger systemic alterations of mIDH1 and mIDH2 characterized by restricted residue flexibility and a reduction in exposure of residues to the solvent surface area. As a result of these structural alterations, crucial interactions of the mutant enzymes were inhibited, a phenomenon that results in a suppression of the production of oncogenic stimulator 2-HG. Findings therefore provide thorough structural and dynamic insights associated with the dual inhibitory activity of AG-881 towards glioma therapy.

Combination olaparib and durvalumab for patients with recurrent IDH-mutated gliomas.

e14026 Background: Isocitrate dehydrogenase (IDH) mutations are frequently observed in low grade gliomas and secondary glioblastoma. Mutant IDH enzymes aberrantly convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG). Accumulation of 2HG inhibits αKG-dependent dioxygenases, many of which are involved in epigenetic regulation. This can lead to cellular dedifferentiation and tumor formation. IDH-mutated cancer cells exhibit defective homologous recombination repair, providing the rationale for investigating poly (adenosine 5’-diphophate-ribose) polymerase (PARP) inhibitors in these tumors. Blockade of programmed cell death ligand 1 (PD‐L1) re‐sensitizes PARP inhibitor treated cancer cells to T‐cell killing. We report the preliminary results of the glioma arm of a clinical trial of a PARP inhibitor, olaparib plus PD-L1 inhibitor, durvalumab, for IDH-mutated solid cancer. Methods: This is a single arm phase II basket study (NCT03991832). Patients with IDH-mutated solid tumors are divided into three cohorts; A: glioma; B: cholangiocarcinoma; C: all other solid tumors. Major eligibility criteria include IDH mutation by immunohistochemistry or sequencing, progressive disease with maximum two prior systemic therapies, Eastern Cooperative Oncology Group performance status (ECOG) 0 –1 and adequate organ function. Patients were excluded if they had received prior PARP inhibitors or anti-PD-1/PD-L1 antibodies. Patients were treated with olaparib 300 mg orally twice daily continuously and durvalumab 1500 mg IV every 4 weeks. Each cycle was 4 weeks. Tumor response was evaluated by MRI after every 2 cycles of study treatments using response evaluation criteria in solid tumors (RECIST). Results: As of Jan 2021, 9 patients were enrolled in Arm A, 7 men and 2 women. The median age was 42 years. Eight patients had IDH1 mutations and 1 had an IDH2 mutation. There were two patients with 1p/19q codeletion. Two patients had grade 2 tumors, four had grade 3, and three had grade 4 tumors. Median time since tumor diagnosis was 7 years. Objective response was seen in 1 patient with an IDH-mutated glioblastoma who remains on study treatments after 8 cycles. Six patients (67%) had tumor progression after two cycles. Two patients had stable disease as per RECIST but had clinical deterioration and did not continue the combined treatment. Common treatment emergent adverse events were all grade 1: fatigue (8 patients), nausea (6), abdominal pain (3), anemia (3), thrombocytopenia (3), and diarrhea (2). Median progression free survival was 2.5 months (range 1.9–8 months). Updated analysis and correlative studies will be presented at the meeting. Conclusions: Combination treatment with olaparib and durvalumab for patients with IDH-mutated glioma is well tolerated but appears to lack adequate antitumor activity. Clinical trial information: NCT03991832.

Phase II basket trial of olaparib and durvalumab in patients (pts) with isocitrate dehydrogenase (IDH) mutated solid tumors.

TPS3167 Background: Somatic IDH mutations are common in low grade gliomas, and rare in other solid tumors with the exception of intrahepatic cholangiocarcinoma (ICA) and certain subtypes of chondrosarcoma. IDH mutations confer a gain-of-function neomorphic activity, such that mutant IDH enzymes preferentially convert αKG to 2-hydroxyglutarate (2HG), resulting in abnormal accumulation of 2HG. 2HG competitively inhibits αKG-dependent dioxygenases, many of which are involved in DNA repair. Preclinical studies show that IDH mutated cancer cells have defective homologous recombination repair and are exquisitely sensitive to poly (adenosine 5’-diphophate-ribose) polymerase (PARP) inhibition. Methods: This is a single arm phase II basket study (NCT03991832). Pts with IDH mutated solid tumors are divided into three cohorts; A: low-grade glioma; B: cholangiocarinoma; C: all other solid tumors. Pts are treated with olaparib 300 mg twice daily continuously and durvalumab 1500 mg every 4 weeks until progression, intolerable toxicity or consent withdrawal. Radiological assessment is performed after every 2 cycles of study treatments. Major eligibility criteria include IDH mutation by immunohistochemistry or sequencing, up to 2 lines of systemic therapy for advanced disease, performance status 0 – 2 and adequate organ function. Pts are excluded if they received prior PARP inhibitors and anti-PD-1/PD-L1 antibody. The Simon’s optimal 2-stage design is applied for Cohorts A and B. 10 pts will be enrolled in each of Cohort A and B initially. If 2 or more partial responses (PR) are seen in these 10 pts, additional 19 pts will be enrolled for a total of 29 pts in that cohort. The combination is considered to be of clinical interest for further development if ≥ 6 PRs are seen in each cohort. Cohort C will enroll 20 pts. Archival tumor tissues and serial blood samples will be collected on study. 2HG levels will be measured and correlated with responses. The study was activated in December 2019. 3 pts have been enrolled into Cohort B. Clinical trial information: NCT03991832 .

AGILE: A Phase 3, Multicenter, Double-Blind, Randomized, Placebo-Controlled Study of Ivosidenib in Combination with Azacitidine in Adult Patients with Previously Untreated Acute Myeloid Leukemia with an IDH1 Mutation

AGILE: A Phase 3, Multicenter, Double-Blind, Randomized, Placebo-Controlled Study of Ivosidenib in Combination with Azacitidine in Adult Patients with Previously Untreated Acute Myeloid Leukemia with an IDH1 Mutation

Abstract 3607: Inhibition of mutant IDH enzymes reduces production of 2-HG but does not restore wild type IDH activity in vitro or in vivo

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in variety of malignancies, and notably >70% of low grade gliomas. These mutations lead to neomorphic enzymatic activity that results in the production of the oncometabolite (D)-2-hydroxyglutarate (2-HG) through the consumption of NADPH. IDH mutant cells and tumors have been shown to have heightened sensitivity to reactive oxygen species (ROS) and radiation, though it is unclear if this sensitivity is due to a loss of IDH wild type enzymatic NADPH production, the neoenzymatic activity of the mutant enzyme, or a combination of these activities. In support of the hypothesis that sensitivity to ROS and radiation is due to a reduced IDH wild type activity in IDH mutant cells, we previously demonstrated that combined treatment of mutant IDH mouse xenografts with AG-881, a potent orally available mutant IDH inhibitor, and radiation resulted in superior tumor growth inhibition than either modality alone. To extend this investigation, we have now developed a novel assay to read out the wild type activity of IDH1/2 enzymes in both cell-based assays and mouse xenograft models. Using an isotopic labeling approach that allows us to fully differentiate the activity of wild type IDH1/2 from the mutant enzymatic activity in the same cell or tumor, we observed that mutant IDH cells and tumors have a significantly reduced level of wild type IDH1/2 activity compared to normal tissue or IDH wild type tumors. Importantly, pharmacologic inhibition of mutant IDH1/2 that resulted in >90% reduction in 2-HG production in mutant cells and tumors did not restore the IDH1/2 wild type activity. These data support our previous observations and suggest that therapeutic strategies targeting the reduced activity of wild type IDH enzymes, such as modalities that induce ROS or treatment with radiation, would be effective when combined with mutant IDH inhibitors. Citation Format: Katie Sellers, Taryn Sleger, Sebastien Ronseaux, Rohini Narayanaswamy, Christine Hudson, Thomas Roddy, Brandon Nicolay. Inhibition of mutant IDH enzymes reduces production of 2-HG but does not restore wild type IDH activity in vitro or in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3607.

Abstract 4956: Functional characterization of the ivosidenib (AG-120) and azacitidine combination in a mutant IDH1 AML cell model

Abstract Approximately 6-10% of patients with acute myeloid leukemia (AML) have a mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1), which leads to production of the oncometabolite 2-hydroxyglutarate (2-HG). Accumulation of 2-HG inhibits α-ketoglutarate-dependent enzymes, resulting in epigenetic dysregulation and a block in cell differentiation, promoting AML. Ivosidenib (IVO; AG-120) is a targeted inhibitor of the mutant IDH1 (mIDH1) enzyme in development for the treatment of AML with an IDH1 mutation. Clinical responses to IVO have been observed in mIDH1 AML, with differentiation of myeloblasts seen in responders indicative of the mechanism of action. Azacitidine (AZA) is a hypomethylating agent that has shown clinical activity in AML. We hypothesized that the combination of IVO + AZA would have a synergistic effect on the release of differentiation block in mIDH1 cells. In an in vitro erythropoetin differentiation assay, human TF-1 erythroleukemia cells expressing the IDH1-R132H mutation (TF1-IDH1R132H) were treated with vehicle, IVO alone (0.2 or 1 µM), AZA alone (0.1, 0.3, or 1 µM), or IVO + AZA concurrently. Assessments were hemoglobinization (red color change), expression of CD235a (erythroid differentiation marker) by flow cytometry, and Kruppel-like factor 1 (KLF1) and hemoglobin gene A/B (HBG) RNA levels by qRT-PCR. Apoptosis was assessed using IncuCyte ZOOM real-time imaging. AZA alone had little or no effect on hemoglobinization, but IVO alone showed dose-dependent increases in hemoglobinization. Concurrent IVO + AZA treatment had a greater effect on hemoglobinization than IVO alone. Increases in CD235a expression were seen with IVO and AZA as single agents, and expression was further increased with the combination of IVO + AZA (at AZA doses ≥0.3 µM). Expression of HBG and KLF1 RNA was not increased by single agent AZA, but was increased by single agent IVO. Concurrent IVO + AZA treatment resulted in potentiation of HBG expression (46-fold increase with IVO [0.2 µM] + AZA [1.0 µM] combination versus 25-fold and 1-fold increases for IVO [0.2 µM] and AZA [1.0 µM] alone, respectively); potentiation of KLF1 expression was less marked. IVO alone had no effect on induction of apoptosis, AZA increased apoptosis versus control, and concurrent treatment with IVO (0.1 and 0.3 µM) + AZA (1 µM) showed greater induction of apoptosis than AZA alone, suggesting potentiation. We have demonstrated that combining IVO + AZA in the TF1-IDH1R132H AML cell line enhanced cell differentiation, as measured by increases in hemoglobinization and expression of differentiation markers, and potentiated cell death, compared with either agent alone. Further exploration of the AZA + IVO combination in AML is ongoing in two clinical trials (NCT03173248 and NCT02677922). Citation Format: Katharine Yen, Vivek S. Chopra, Erica Tobin, Brian Avanzino, Konstantinos Mavrommatis, Jorge DiMartino, Kyle J. MacBeth. Functional characterization of the ivosidenib (AG-120) and azacitidine combination in a mutant IDH1 AML cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4956.

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79 ± 6% yield (n = 9), and 18F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ± 1.6% (n = 5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with 18F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.

Abstract A071: AG-120 (ivosidenib), a first-in-class mutant IDH1 inhibitor, promotes morphologic changes and upregulates liver-specific genes in IDH1 mutant cholangiocarcinoma

Abstract Background: Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been reported in ~25% of patients with intrahepatic cholangiocarcinoma (CC). The mutant IDH1 (mIDH1) enzyme has a gain-of-function activity, catalyzing the reduction of alpha-ketoglutarate to D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG leads to epigenetic dysregulation, and potential silencing of genes that control cellular differentiation. In a preclinical model of liver development, IDH1 mutation resulted in HNF4A silencing and impaired hepatocyte differentiation. AG-120 (ivosidenib) is a first-in-class, oral, selective inhibitor of mIDH1 that has been shown to reduce 2-HG levels in plasma and tumor tissue in preclinical and clinical studies, and to induce differentiation of leukemic blasts in patients with acute myeloid leukemia. In solid tumors, the consequences of IDH1 mutations and the effects of inhibition are less understood. In AG120-C-002 (ClinicalTrials.gov NCT02073994), a phase 1 clinical trial in mIDH1 advanced solid tumors, 73 patients with CC were treated with AG-120 at doses ranging from 100 mg twice daily to 1200 mg once daily. Among the phase 1 CC subjects who underwent on-study biopsies, we assessed morphologic and gene expression profile changes to understand the relationship between these parameters. Methods: Biopsy samples were collected from patients with CC enrolled in AG120-C-002 at screening, Cycle 3 Day 1, and in some cases Cycle 7 Day 1, disease progression, and end of treatment. Fourteen pre- and post-dose formalin-fixed paraffin-embedded (FFPE) tumor sample pairs were evaluable for morphology assessment (from 27 FFPE pre- and post-dose pairs collected), and 19 pre- and post-dose fresh frozen tumor sample pairs underwent gene expression profiling (from 38 pre- and post-dose pairs collected). Both morphology and gene expression data were assessed in eight patients to elucidate their relationship. Hematoxylin and eosin stained sections were evaluated for a cholangiocellular growth pattern and cytoplasmic alterations by two gastrointestinal pathologists from independent institutions. Gene expression profiles were generated by RNA sequencing to relate to morphologic changes. Results: Morphologic assessment of 14 pre- and post-dose sample pairs showed that upon 8-24 weeks of AG-120 treatment, a subset of CCs exhibited change in two phenotypes: (a) an increase of 20% or more in the cholangiocellular growth pattern (n=5), and (b) a reduction in the volume of cytoplasm (n=8). Changes in both phenotypes (a) and (b) were seen in five cases. RNA sequence profiling in samples with increased cholangiolar histology also showed a trend toward increased liver-specific gene expression. Conclusions: This is the first demonstration that AG-120 treatment may induce morphologic and molecular changes in a subset of mIDH1 CCs. Correlation of changes in morphology and gene expression with specific clinical outcomes will be described. Further studies, such as methylation changes, protein change by immunohistochemistry, and preclinical studies, are warranted to understand the biology of these morphologic and gene expression changes. Citation Format: Yuko Ishii, Carlie Sigel, Maeve A. Lowery, Lipika Goyal, Camelia Gliser, Liewen Jiang, Susan Pandya, Bin Wu, Sung Choe, Vikram Deshpande. AG-120 (ivosidenib), a first-in-class mutant IDH1 inhibitor, promotes morphologic changes and upregulates liver-specific genes in IDH1 mutant cholangiocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A071.

Phase III randomized, open-label study comparing the efficacy and safety of AG-221 vs conventional care regimens (CCR) in older patients with advanced acute myeloid leukemia (AML) with isocitrate dehydrogenase (IDH)-2 mutations in relapse or refractory to multiple prior treatments: the IDHENTIFY trial.

TPS7074 Background: Mutant IDH (mIDH) enzymes produce an oncometabolite, 2-hydroxyglutarate (2HG), that can prevent DNA and histone demethylation required for lineage-specific myeloid progenitors to differentiate into mature cells. mIDH2 occurs in 8-19% of AML patients (pts) (Döhner, NEJM 2015) and can survive chemotherapy, leading to relapse (Chan, Nature Med 2015). AG-221 is a first in class, potent, selective, oral inhibitor of mIDH2. In a phase 1 dose-finding/expansion study of AG-221 in 159 pts with mIDH2+ AML relapsed-refractory (R/R) after 1 to 6 prior AML treatment (Tx) regimens, hematologic response rate was 38%, including 18% complete remission rate (Stein, ASH 2015). The international IDHENTIFY study (NCT02577406) in pts with mIDH2+ AML is the first randomized trial to evaluate an AML Tx in older pts R/R to multiple prior Tx courses—a pt population with few Tx options. Methods: Eligible pts are age ≥60 years with primary or secondary mIDH2+ AML and R/R after 2 or 3 prior Tx regimens. Physicians preselect the most appropriate of 4 CCR, given in 28-day cycles, for each pt (Table). Pts are then randomized 1:1 to AG-221 or to CCR and receive the preselected Tx. Randomization is stratified (yes/no) by prior intensive chemotherapy, primary refractory, and prior allogeneic HSCT. Assuming median OS of 5 months with CCR (Roboz, JCO, 2014) and 8 months with AG-221 (60% improvement) and a drop-out rate of ~10%, 280 pts will allow enough events (193 deaths) to achieve ≥90% power to detect a statistically significant OS difference at a Type I error rate of 0.05 (two-sided). Enrollment began in Oct. 2015 and continues through 2019. Clinical trial information: NCT02577406.Tx Arm* Dose Endpoints CCR 75 mg/m2 SC QD x7d Primary: Overall Survival (OS) Azacitidine 20 mg SC BID x10d Secondary: Overall response rate (IWG AML criteria), event-free survival, time to and duration of response, 1-year survival, quality of life Low-dose ara-C 0.5-1.5 g/m2 IV QD x3-6d Intermediate-dose ara-C 100 mg PO QD x28d Best supportive care (BSC) only AG-221

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs

IntroductionMalignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). MethodsA small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine (125I) and/or 18F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [125I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. ResultsEnzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [125I]1 (n = 4) and 67.5 ± 6.6% for [18F]4 (n = 3). [125I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [125I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. ConclusionsThese data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

The Role of IDH Mutants, Which Are Promising Therapeutic Targets for Acute Myeloid Leukemia

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents.To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML.Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such as Ebf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered by conditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions of these genes are downregulated in the AML cells and increased by deletion of IDH2/R140Q.Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN that hydroxylates and marks HIF1α for ubiquitin-proteasomal degradation, it is probable that mutant IDH2-produced 2HG stabilizes HIF1α through inhibition of EGLN. Furthermore, it was reported that Meis1 activates the transcription of HIF1α. In consistent with these information, we showed that IDH2/R140Q increased the protein levels of HIF1α in cultured cells. DisclosuresMatsunaga:Daiichi Sankyo Co., Ltd.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Abstract LB-252: IDH mutations are promising targets for acute myeloid leukemia

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that IDH2/R140Q decreased the level of 5hmC and the expression of differentiation-inducing genes, including Ebf1, Spib and Pax5. Gene expression analysis revealed that IDH2/R140Q activated the hypoxia pathway and the expression of Meis1. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Citation Format: Yoko Ogawara, Hironori Matsunaga, Takahiko Seki, Yukino Machida, Kazushi Araki, Issay Kitabayashi. IDH mutations are promising targets for acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-252. doi:10.1158/1538-7445.AM2015-LB-252

IDH mutations activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia model mice

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs mediate exchanges between isocitrate and α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which causes dysregulation of a set of α-KG-dependent dioxygenases such as TET, histone demethylase and others. Because mutant IDH has no necessary functions in normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH2-dependent AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted mutant IDH2 from AML mice, which resulted in the loss of leukemia stem cells and significantly delayed the progression of AML. These results indicate that mutant IDHs are promising targets for anticancer therapy.