Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

PPM1D Is a Therapeutic Target in Childhood Neural Tumors

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

Navtemadlin (KRT-232), a Small Molecule MDM2 Inhibitor, Is More Effective Than Decitabine Against Myeloproliferative Neoplasm-Blast Phase in a Patient-Derived Xenograft Model

Patients with myeloproliferative neoplasm-blast phase (MPN-BP) have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies (Mesa Blood 2015). Decitabine is the standard of care for MPN-BP. Recently, using xenotransplantation assays, we have shown that MPN-BP originates at the hematopoietic stem cell (SC) level (Wang Blood 2018) and that MPN-BP CD34 + cells contain higher levels of MDM2 protein compared with their normal counterparts (% of MDM2 + CD34 + cells: MPN-BP: 76.4±3.3; Normal: 17.5±6.4. P <0.05). MDM2 negatively regulates p53 activity and MDM2 inhibitors can activate p53 and induce apoptosis of TP53 WT cancer cells. As mutations or deletions of TP53 occur infrequently in MPN-BP, we examined the effects of a potent MDM2 inhibitor, navtemadlin (KRT-232; Canon Mol. Cancer Ther. 2015) and decitabine as monotherapy or in combination on the depletion or elimination of MPN-BP cells in a patient derived xenograft (PDX) model. We firstestablished the dynamics of leukemia cell recovery following a single cycle of navtemadlin treatment. Spleen cells with wild type (WT) TP53 and mutations in KRAS, RAD21, KMT2A and ASXL1 were collected from the 4 th generation of MPN-BP PDX mice. Forty days after injection of these spleen cells (4×10 5/mouse) into sublethally irradiated NSG mice, peripheral blood (PB) leukemic burden (hCD34 + cells: 0.39±0.09%) was demonstrated by flow cytometric analysis. Mice were treated with vehicle alone (n=3) or high doses of navtemadlin (100 mg/kg, n=4) by daily oral gavage on day (D) 1-7. Without navtemadlin treatment, leukemia engraftment and leukemic burden continued to increase until the mice died on D15-D23. By contrast, hCD34 + leukemic blasts were almost undetectable on D8 and remained at significantly lower levels in PB of mice treated with navtemadlin on D15, than were detected in mice receiving vehicle alone (D8: Vehicle: 1.8±1.3%; Navtemadlin: 0.1±0.1%. D15: Vehicle: 19.9±6.0%; Navtemadlin: 0.5±0.1%). These findings suggest that navtemadlin monotherapy has the potential to deplete MPN-BP blast cells and prolong survival in MPN-BP PDX mice. To achieve long-term remission and to prevent relapse, we treated MPN-BP PDX mice with multiple cycles of low (50 mg/kg, n=4) and high dose navtemadlin (100 mg/kg, n=5) at three-week intervals based on the dynamics of leukemia cell recovery following a single cycle of navtemadlin treatment. hCD34 + cells, which contain MPN-BP SCs, and hCD45 dimCD33 + leukemic blasts were reduced in the spleen, but not in the marrows of mice during 3 cycles of 100 mg/kg navtemadlin treatment. However, reduced leukemia cell burden in PB persisted during 3 cycles of navtemadlin treatment and was associated with a prolongation in survival, which was dose-dependent (Mean survival after transplantation: Vehicle: 64.0 days; 50mg/kg navtemadlin: 86.0 days; 100mg/kg navtemadlin: 98.3 days). We then examined if a combination of navtemadlin and decitabine is more effective than single agent therapy in depleting MPN-BP SCs. Again, navtemadlin at 100 mg/kg significantly reduced the leukemia cell burden in mouse PB on C1D8 (hCD34 + cells: Vehicle: 4.1±0.8%; Navtemadlin: 0.6±0.2%, an 85% decrease. hCD45 dimCD33 + cells: Vehicle: 1.4±0.3%; Navtemadlin: 0.04±0.03%, a 97% decrease. P <0.001 for both), which persisted during 2 cycles of treatment. By contrast, multiple cycles of decitabine monotherapy (2.5mg/kg, IP, 3 times/week in 21-day cycles) resulted in a modest reduction in hCD34 + cells and hCD45 dimCD33 + cells (11.4% and 30.1% decrease, respectively) in PB of MPN-BP PDX mice, and did not reduce these cells in either the spleen or bone marrow on C1D8. Finally, addition of decitabine to navtemadlin did not further deplete either MPN-BP SCs or leukemia cells and did not improve survival of MPN-BP PDX mice, as compared to treatment with navtemadlin alone. Mean survival after transplantation in the combination study was: Vehicle: 55.5 days; navtemadlin: 70.3 days; Decitabine: 56.3 days; Combination: 64.0 days. Furthermore, toxicity (body weight loss, intestinal pathology) was observed in mice receiving high dose of navtemadlin and decitabine simultaneously, but not when either drug was administered alone. In conclusion, navtemadlin monotherapy, which activates p53, depletes leukemia cell counts and prolongs survival of MPN-BP PDX mice and is a promising agent for patients with WT TP53 MPN-BP. Disclosures Krejsa: Kartos Therapeutics, Inc.: Current Employment. Hoffman: AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding.

Inhibition of MDM2 Improves the Therapeutic Effect of Hypomethylating Agents in Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML)

Hypomethylating agents (HMA) are the current standard care for MDS and CMML. However, a large proportion of patients experience HMA treatment failure that is associated with poor prognosis. Novel treatments are urgently needed for these diseases. We performed next generation sequencing (NGS) based mutation profiling that targeted the 81 most frequently mutated genes in baseline bone marrow (BM) mononuclear cells (MNCs) from patients with MDS and CMML (N=83) prior to HMA treatment. Analysis of impact of the mutations on outcomes of HMA treatment indicated that patients with TP53 mutations (N=22) had significantly worse overall survival (HR = 3.29; P < 0.05; q < 0.05). This result is consistent with previous reports and suggests that strengthening the activity of wild type (WT) TP53, through strategies such as inhibition of TP53 negative regulator MDM2, may improve therapeutic efficacy of HMAs in patients with MDS and CMML. To test this hypothesis, we evaluated in vitro the effects of the combination of the MDM2 inhibitor DS-3032b and azacytidine (AZA). Due to the dearth of MDS or CMML cell lines, initial analysis was performed in the TP53 WT AML cell line MOLM13. Results indicated that DS-3032b and AZA combination induced a synergistic decrease in cell survival (coefficient of drug interaction=0.65), accompanied with increased apoptosis and CDKN1A1 (P21) RNA expression. No combination survival effect was observed in the TP53 mutant AML cell lines SKM1 or TF1. We next evaluated in vivo combination therapeutic effects of the MDM2 inhibitor DS-5272 and AZA in a Tet2-knockout (KO) (Tet2flox/flox/Vav-Cre) mouse model. Prior work indicated that Tet2-KO mice had an MDS/CMML-like phenotype and a persistent long-term (LT) BM repopulating activity that was resistant to AZA. DS-5272 and AZA were administered to mice sequentially: DS-5272 (50 mg/kg) was administered orally for 5 days, followed by daily AZA (2.5 mg/kg, i.p.) for a 7 days. DS-5272 was also administered on the 1st and 3rd days during AZA treatment. After a 2-week gap, the treatment cycle was repeated once. Following the combination treatment, Tet2-KO mice showed significantly reduced monocytosis (P<0.05) and increased platelet counts (P<0.001) in peripheral blood (PB), and significant reductions in Gr1+/CD11b1+ myeloid cells (P<0.001), common myeloid progenitors (CMPs) (P<0.005), and Lin-/Sca1+/c-Kit1+ cells (LSKs) (P<0.01) in BM. Next, we evaluated the impact of treatment on LT BM repopulation activity. CD45.2+ BM cells isolated from drug-treated Tet2-KO mice were mixed with BM cells from CD45.1 WT mice and then transplanted into lethally irradiated CD45.1 WT recipient mice. Significantly lower (P<0.05) CD45.2 chimerism was observed in PB of the recipients transplanted by BM cells from DS-5272 and AZA combination treated Tet2-KO mice compared to recipients that received cells from vehicle control and mono-agent treated Tet2-KO mice, starting from 4 months post-transplantation (Fig.1A). Lower CD45.2 chimerism was also observed in the BM LSK population (P<0.001) of the same recipients at the endpoint of 6 months post-transplantation. To study the molecular mechanisms underlying the combinational therapeutic effects of the MDM2 inhibitor and AZA observed in the Tet2-KO mice, we performed RNA-seq on BM LSK cells isolated from drug-treated mice. Compared to LSKs from vehicle control mice, over 800 genes with significantly altered RNA expression (>1.5 fold; FDR<0.05) were identified in the LSKs from DS-5272 and AZA combination treated Tet2-KO mice, whereas 110 and 476 genes with altered RNA expression were identified in the LSKs of DS-5272 or AZA treated Tet2-KO mice respectively. REACTOME pathway analysis identified 263 pathways that were significantly (FDR<0.25) and uniquely activated in the BM LSKs from mice treated with DS-5272 and AZA combination. Many of these pathways were associated with TP53 stability, TP53 activity, and TP53 regulated apoptosis and cell cycle (Fig.1B), suggesting a coordination between MDM2 inhibition and HMA treatment to activate TP53 in BM HSPCs. In conclusion, our work provides proof-of-concept evidence that combining an MDM2 inhibitor with AZA can improve the therapeutic efficacy of AZA in MDS and CMML, through mechanisms including synergistic activation of TP53 in BM HSPCs and inhibition of LT BM repopulating activity. This concept should be further evaluated through pre-clinical studies and clinical trials. Figure 1 Figure 1. Disclosures Wei: Daiichi Sanko: Research Funding. Daver: Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Hanmi: Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Genentech: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding.

An Enterohepatic Recirculation Population Pharmacokinetic and MIC-1 Pharmacokinetic-Pharmacodynamic Model for the MDM2 Inhibitor Navtemadlin (KRT-232) in Healthy Subjects

Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent and selective, orally available MDM2 inhibitor restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies. Navtemadlin is currently being evaluated in a phase 3 trial of patients with relapsed or refractory myelofibrosis, as well as in numerous phase 1b/2 trials in various hematologic malignancies and solid tumors. Serum macrophage inhibitor cytokine-1 (MIC-1) is a pharmacodynamic (PD) marker of p53-mediated activity in patients treated with navtemadlin (Allard et al. HemaSphere. 2020). Using pharmacokinetic (PK) and PD data from a healthy subject food effect study (Wong et al. Blood. 2020), we developed a population PK (PPK) model that characterized enterohepatic recirculation (EHR) as a half-life extending element in the PK profiles of navtemadlin and its major acyl glucuronide metabolite M1. MIC-1 PD data were incorporated into the model to quantify plasma concentration-driven MIC-1 excursions and to simulate PK and PD across time and dose in healthy subjects. Methods: PPK and PK-PD models were developed using the first-order conditional estimation with interaction (FOCE-I) method in NONMEM 7.4, with model covariates selected using a stepwise forward addition and backward elimination method based on a 5% significance level. Model quality was checked by inspecting model parameters and confidence intervals, as well as standard residual-based and simulation-based diagnostics, and prediction-corrected visual predictive checks. Navtemadlin plasma concentration and MIC-1 serum concentration-time data from the food effect study (KRT-232-105) were modeled (N=30 subjects after a single 60 mg navtemadlin dose). Candidate PPK semi-mechanistic models that described EHR with multi-compartment structures (gut, central, and peripheral compartments for navtemadlin, and central and gallbladder [GB] compartments for M1), first-order elimination, and mealtime effects on GB emptying were tested. Post hoc parameter estimates from the final PPK model were used to generate individual predicted navtemadlin plasma concentrations for the PK-PD model. Based on exploratory plots, the pharmacological mechanism of action of navtemadlin, and a bile acid recycling model (Guiastrennec et al. CPT Pharmacometrics Syst Pharmacol. 2018), an indirect response equation was selected for the MIC-1 effect compartment (Figure 1a). Results: Navtemadlin and M1 plasma concentrations, including a second peak attributed to EHR at ~8-12 h, were well described by a model with central and peripheral compartments, constant basal M1 release rate into bile (KBR BASAL), and incremental mealtime GB emptying rate (KBR MEAL, Figure 1a). Figure 1b shows simulated navtemadlin and M1 amounts in various compartments over time. Median oral clearance of navtemadlin was estimated at 36.35 L/h. Estimated median apparent oral clearance of navtemadlin in healthy subjects was higher than PPK estimates for patients with advanced solid tumors (24.9 L/h [Ma et al. Blood. 2019]). The median central and peripheral volumes of navtemadlin were 159 L and 390 L, respectively. Navtemadlin exposure was higher in healthy female subjects relative to male subjects. Between-subject variability in clearance was 31%. Typical MIC-1 maximum stimulatory effect (S max) was estimated at 6.82, close to the median maximum ratio of MIC-1 to baseline MIC-1 (7.29) in the observed data. SC 50 was estimated at 85.22 ng/mL, with a Hill coefficient of 2.02, indicating a relatively steep increase in MIC-1 serum concentration with increasing navtemadlin concentration. For both PPK and PK-PD models, diagnostic plots confirmed an adequate fit. Subjects with lower baseline MIC-1 had a larger response and reached a maximum MIC-1 concentration later. Older subjects had the largest covariate impact, with a higher MIC-1 response. Conclusion: A two-compartment PPK model with basal and incremental mealtime GB emptying rates captured concentration-time data for navtemadlin and its metabolite M1. EHR was evident and navtemadlin reabsorption following hydrolysis of biliary M1 in the intestine contributed to navtemadlin half-life. An indirect stimulatory PK-PD model effectively described the relationship between navtemadlin and MIC-1 in healthy subjects. Figure 1 Figure 1. Disclosures Zhang: Certara, Inc.: Current Employment; Milad Pharmaceutical Consulting, LLC.: Ended employment in the past 24 months. Wong: Kartos Therapeutics: Current Employment; AbbVie Biotherapeutics: Current equity holder in publicly-traded company. Slatter: Telios Pharma: Current holder of stock options in a privately-held company; Kartos Therapeutics: Current Employment, Current holder of stock options in a privately-held company; AstraZeneca: Current equity holder in publicly-traded company; Amgen: Divested equity in a private or publicly-traded company in the past 24 months. OffLabel Disclosure: Yes, navtemadlin (KRT-232) is an investigational small molecule MDM2 inhibitor.