P53 Wild-type Acute Myeloid Leukemia Research Articles

KT-253, A Novel MDM2 Degrader and p53 Stabilizer, Has Superior Potency and Efficacy Than MDM2 Small Molecule Inhibitors.

Murine double minute 2 (MDM2) is an E3 ligase that inhibits the tumor suppressor protein p53. Clinical trials employing small-molecule MDM2/p53 interaction inhibitors (SMIs) have demonstrated limited activity, underscoring an unmet need for a better approach to target MDM2. KT 253 is a highly potent and selective heterobifunctional degrader that overcomes the MDM2 feedback loop seen with SMIs and induces apoptosis in a range of hematologic and solid tumor lines. A single intravenous dose of KT 253 triggered rapid apoptosis and sustained tumor regression in p53 wild-type acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) xenograft models. Additionally, a single intravenous dose of KT 253 in combination with standard-of-care (SoC) venetoclax, overcame venetoclax resistance in an AML xenograft model. The data herein define the therapeutic potential of KT-253 and support its clinical development in a range of hematologic and solid p53 wild-type (WT) malignancies, as a monotherapy and in combination with SoC agents.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Preliminary Results from a Phase Ib Study Exploring MDM2 Inhibitor Siremadlin (HDM201) in Combination with B-Cell Lymphoma-2 (BCL-2) Inhibitor Venetoclax in Patients with Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (HR-MDS)

Preliminary Results from a Phase Ib Study Exploring MDM2 Inhibitor Siremadlin (HDM201) in Combination with B-Cell Lymphoma-2 (BCL-2) Inhibitor Venetoclax in Patients with Acute Myeloid Leukemia (AML) or High-Risk Myelodysplastic Syndrome (HR-MDS)

Abstract 4830: Selective DNA-PK inhibitor, M3814, boosts p53 apoptotic response to DNA double strand breaks and effectively kills acute leukemia cells: Implications for AML therapy

Abstract M3814 is a selective inhibitor of DNA-PK in clinical development. It effectively blocks non-homologous end joining repair of DNA double strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By inhibiting DNA-PK catalytic activity in the presence of DSB damage, M3814 disrupts a previously unrecognized negative regulatory loop between DNA-PK and ATM, leading to increased activation of the ATM pathway, including its downstream targets, CHK2 and p53. As a result, in cancer cells expressing wild-type p53, DNA-PK inhibitor strongly activates the p53 response to DSB DNA damage and its two main functions- cell cycle arrest and apoptosis. While p53 activation in p53 wild-type epithelial cancer cells causes effective cell cycle arrest, the majority of the epithelial tumors lose their ability to undergo p53 dependent apoptosis. In contrast, tumor cells from patients with acute leukemia express wild-type p53 in with preserved p53 apoptotic function, making them amenable to p53 activation approaches. Here, we investigate the therapeutic potential of M3814 in combination with DSB-inducing agents in acute leukemia cells. M3814 strongly potentiated the activity of IR in three p53 wild-type leukemia cell lines (MOLM-13, MOLT-4, MV4-11) but had minimal effect on the p53-null HL-60 and THP-1 lines. This potentiation was due to enhanced activation of the p53 pathway as demonstrated by substantially increased expression of p53 transcriptional targets, p21 and PUMA, and induction of apoptosis only in p53 wild-type cells. M3814 showed synergism in p53-dependent cell killing with the topoisomerase inhibitors, daunorubicin and idarubicin, and 2-3 fold increase in the potency of daunorubicin/cytarabine combination in p53 wild-type acute myeloid leukemia (AML) cells. Daunorubicin/cytarabine/M3814 combinations were better tolerated by CD34+ bone marrow cells harvested from healthy volunteers and tested in vitro. These results support further exploration of M3814 as a novel combination partner in the treatment of p53 wild-type AML. Citation Format: Eric Haines, Astrid Zimmermann, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. Selective DNA-PK inhibitor, M3814, boosts p53 apoptotic response to DNA double strand breaks and effectively kills acute leukemia cells: Implications for AML therapy [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 4830.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34+ AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53mut), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53R273H. AML CD34+ cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34+ cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53wt), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53mut. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34+ cells were separated into ROSlow and ROShigh subfractions. The immature AML CD34+-enriched ROSlow cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROShigh cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34+ cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

BackgroundVenetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML.MethodsThe effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments.ResultsCombination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies.ConclusionsOur findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0280-3) contains supplementary material, which is available to authorized users.

Selective induction of TP53I3/p53-inducible gene 3 (PIG3) in myeloid leukemic cells, but not in normal cells, by Nutlin-3

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 is a promising anti-cancer agent, which exhibits activity against a variety of cancers, including acute myeloid leukemia (AML). Previous studies have shown that Nutlin-3 variably induces apoptosis and cell cycle arrest in cancer cells while it shows low/absent cytotoxicity in normal cells. However, the reason for the selective pro-apoptotic activity in cancer cells with respect to normal counterparts is incompletely understood. In this study, we have compared the induction of several known target genes of p53 in two p53(wild-type) AML cell lines, OCI-AML3 and MOLM, in comparison with primary normal peripheral blood mononuclear cells (PBMC). Among several p53-target genes activated both in AML cell lines and normal PBMC (BBC3, BAX, MDM2, FAS, CDKN1A, GDF15, GADD45A, TNFRSF10B, TP53I3/PIG3), only TP53I3/PIG3 was selectively activated in MOLM and OCI-AML3, but not in PBMC. The important role of TP53I3/PIG3 in mediating the apoptotic activity of Nutlin-3 was underlined by knock-down experiments with siRNA specific for TP53I3/PIG3, which resulted in a significant decrease in the pro-apoptotic activity of Nutlin-3.

The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2 inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML.

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.