MDM2 antagonist idasanutlin in patients with polycythemia vera: results from a single-arm phase 2 study

Idasanutlin, an MDM2 antagonist, showed clinical activity and rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label, phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily, days 1-5 of each 28-day cycle. The primary endpoint was composite response (hematocrit control and spleen volume reduction >35%) in patients with splenomegaly, and hematocrit control in patients without splenomegaly at week 32. Key secondary endpoints included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n=27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n=13), 9 (69%) attained any spleen volume reduction and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (n=6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥3 nausea and vomiting experienced in 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. Registration: NCT03287245.

Phase I study of daily and weekly regimens of the orally administered MDM2 antagonist idasanutlin in patients with advanced tumors

Summary Aim The oral MDM2 antagonist idasanutlin inhibits the p53-MDM2 interaction, enabling p53 activation, tumor growth inhibition, and increased survival in xenograft models. Methods We conducted a Phase I study of idasanutlin (microprecipitate bulk powder formulation) to determine the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, food effect, and clinical activity in patients with advanced malignancies. Schedules investigated were once weekly for 3 weeks (QW × 3), once daily for 3 days (QD × 3), or QD × 5 every 28 days. We also analyzed p53 activation and the anti-proliferative effects of idasanutlin. Results The dose-escalation phase included 85 patients (QW × 3, n = 36; QD × 3, n = 15; QD × 5, n = 34). Daily MTD was 3200 mg (QW × 3), 1000 mg (QD × 3), and 500 mg (QD × 5). Most common adverse events were diarrhea, nausea/vomiting, decreased appetite, and thrombocytopenia. Dose-limiting toxicities were nausea/vomiting and myelosuppression; myelosuppression was more frequent with QD dosing and associated with pharmacokinetic exposure. Idasanutlin exposure was approximately dose proportional at low doses, but less than dose proportional at > 600 mg. Although inter-patient variability in exposure was high with all regimens, cumulative idasanutlin exposure over the whole 28-day cycle was greatest with a QD × 5 regimen. No major food effect on pharmacokinetic exposure occurred. MIC-1 levels were higher with QD dosing, increasing in an exposure-dependent manner. Best response was stable disease in 30.6% of patients, prolonged (> 600 days) in 2 patients with sarcoma. Conclusions Idasanutlin demonstrated dose- and schedule-dependent p53 activation with durable disease stabilization in some patients. Based on these findings, the QD × 5 schedule was selected for further development. Trial registration NCT01462175 (ClinicalTrials.gov), October 31, 2011.

Pharmacological Activation of p53 during Human Monocyte to Macrophage Differentiation Attenuates Their Pro-Inflammatory Activation by TLR4, TLR7 and TLR8 Agonists

The transcription factor p53 has well-recognized roles in regulating cell cycle, DNA damage repair, cell death, and metabolism. It is an important tumor suppressor and pharmacological activation of p53 by interrupting its interaction with the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2) is actively explored for anti-tumor therapies. In immune cells, p53 modulates inflammatory responses, but the impact of p53 on macrophages remains incompletely understood. In this study, we used the MDM2 antagonist idasanutlin (RG7388) to investigate the responses of primary human macrophages to pharmacological p53 activation. Idasanutlin induced a robust p53-dependent transcriptional signature in macrophages, including several pro-apoptotic genes. However, idasanutlin did not generally sensitize macrophages to apoptosis, except for an enhanced response to a Fas-stimulating antibody. In fully differentiated macrophages, idasanutlin did not affect pro-inflammatory gene expression induced by toll-like receptor 4 (TLR4), TLR3, and TLR7/8 agonists, but inhibited interleukin-4-induced macrophage polarization. However, when present during monocyte to macrophage differentiation, idasanutlin attenuated inflammatory responses towards activation of TLR4 and TLR7/8 by low doses of lipopolysaccharide or resiquimod (R848). This was accompanied by a reduced expression of CD14, TLR7, and TLR8 in macrophages differentiated in the presence of idasanutlin. Our data suggest anti-inflammatory effects of pharmacological p53 activation in differentiating human macrophages.

Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

Novel Therapeutic Insights in Dedifferentiated Liposarcoma: A Role for FGFR and MDM2 Dual Targeting

We aimed to evaluate the therapeutic potential of the pan-FGFR inhibitor erdafitinib to treat dedifferentiated liposarcoma (DDLPS). FGFR expression and their prognostic value were assessed in a series of 694 samples of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). The effect of erdafitinib—alone or in combination with other antagonists—on tumorigenicity was evaluated in vitro and in vivo. We detected overexpression of FGFR1 and/or FGFR4 in a subset of WDLPS and DDLPS and demonstrated correlation of this expression with poor prognosis. Erdafitinib treatment reduced cell viability, inducing apoptosis and strong inhibition of the ERK1/2 pathway. Combining erdafitinib with the MDM2 antagonist RG7388 exerted a synergistic effect on viability, apoptosis, and clonogenicity in one WDLPS and two DDLPS cell lines. Efficacy of this combination was confirmed in vivo on a DDLPS xenograft. Importantly, we report the efficacy of erdafitinib in one patient with refractory DDLPS showing disease stabilization for 12 weeks. We provide evidence that the FGFR pathway has therapeutic potential for a subset of DDLPS and that an FGFR1/FGFR4 expression might constitute a powerful biomarker to select patients for FGFR inhibitor clinical trials. In addition, we show that combining erdafitinib with RG7388 is a promising strategy for patients with DDLPS that deserves further investigation in the clinical setting.

MDM2 Antagonists Induce a Paradoxical Activation of Erk1/2 through a P53-Dependent Mechanism in Dedifferentiated Liposarcomas: Implications for Combinatorial Strategies

The MDM2 gene is amplified in dedifferentiated liposarcoma (DDLPS). Treatment with MDM2 antagonists is a promising strategy to treat DDLPS; however, drug resistance is a major limitation when these drugs are used as a single agent. This study examined the impact of MDM2 antagonists on the mitogen-activated protein kinase (MAPK) pathway in DDLPS and investigated the potential synergistic activity of a MAPK kinase (MEK) inhibitor in combination with MDM2 antagonists. We identified a synergistic effect and identified the mechanism behind it. Combination effects of MDM2 antagonists and a MEK inhibitor were analyzed in a patient-derived xenograft mouse model and in DDLPS and leiomyosarcoma cell lines using different cell proliferation assays and immunoblot analysis. MDM2 antagonist (RG7388)-resistant IB115 [P4] cells and p53-silenced DDLPS cells were also established to understand the importance of functional p53. We found that MDM2 antagonists induced an upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK) in DDLPS cells. The upregulation of p-ERK occurred due to mitochondrial translocation of p53, which resulted in increased production of reactive oxygen species, causing the activation of receptor tyrosine kinases (RTKs). Activated RTKs led to the activation of the downstream MEK/ERK signaling pathway. Treatment with a MEK inhibitor resulted in decreased expression of p-ERK, causing significant anti-tumor synergy when combined with MDM2 antagonists. Our results provide a framework for designing clinical studies of combination therapies in DDLPS patients.