PF337 ALRN‐6924, A DUAL INHIBITOR OF MDMX AND MDM2 THAT CAUSES MINIMAL THROMBOCYTOPENIA IN PATIENTS, DISRUPTS DIFFERENT STAGES OF THROMBOPOIESIS COMPARED TO MDM2‐ONLY INHIBITION
Authors: Carvajal L. , Ren J.‐G. , Guerlavais V. , Steidl U. , Aivado M. , Annis A. 
Published: August 2019

Background:

ALRN‐6924 is a cell‐permeating α‐helical peptide that disrupts interactions of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. ALRN‐6924 can thereby restore p53‐dependent anti‐cancer activity in p53 wild type tumors. In an ongoing ALRN‐6924 clinical trial with 96 solid tumor and lymphoma patients enrolled as of 13Aug2018 there have been 1/0 adverse events (AE) of Grade 3/4 thrombocytopenia (TCP), respectively, at doses that yield objective responses (NCT02264613; Meric‐Bernstam et al, ASCO 2017; Shustov et al, ASH 2018). In contrast, small molecule MDM2‐only inhibitors induce Grade 3–4 TCP as a common AE and dose‐limiting toxicity.

Aims:

To explore the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 and MDM2i's, we investigated the effects of dual MDM2/MDMX‐inhibition vs. MDM2‐only inhibition during different stages of megakaryocyte maturation.

Methods:

Clonogenicity was measured in megakaryocyte (MK), erythroid (E), and granulocyte/monocyte myeloid (GM) progenitor colony‐forming unit (CFU) assays. Human bone marrow CD34+ cells seeded in methylcellulose semi‐solid media containing IL‐3 and IL‐6; and TPO, Epo or GM‐CSF together with ALRN‐6924, RG7388 (idasanutlin), or controls. CFU‐MK, CFU‐E, and CFU‐GM colonies were measured after 14 days of culture. Megakaryocyte maturation was modeled in vitro using a 14‐day liquid culture assay. Human bone marrow CD34+ cells were cultured in liquid medium containing SCF and TPO. Megakaryocyte maturation was assessed by measuring the expression of CD41 and CD42. To assess the effects of ALRN‐6924 and RG7388 on platelet development, drugs were added to the culture at different stages during the 14‐day period of megakaryocyte maturation in vitro. DNA synthesis was measured by BrdU, and apoptosis by Annexin V/propidium iodide. Healthy C57BL/6 mice treated with ALRN‐6924 3x/week were monitored for blood counts 2x/week; hematopoietic stem and progenitor cells in bone marrow were measured at 4 weeks.

Results:

In vitro, ALRN‐6924 disrupted MK colony formation more potently than RG7388, with IC50's of 0.024 and 0.12 μM, respectively, while their effects were comparable in CFU‐E and CFU‐GM assays with IC50's for both of ≈ 0.2 μM. Conversely, RG7388 disrupted megakaryopoiesis by inducing apoptosis and cell cycle arrest in maturing MKs 10‐fold more potently than ALRN‐6924. Consistent with these findings, ALRN‐6924 induced minimal effects on platelet counts in mice at doses that are effective in xenotransplanted and syngeneic tumor models, despite a significant decrease in MK and erythroid progenitors, while more immature hematopoietic stem and progenitor cells numbers remained unperturbed.

Summary/Conclusion:

ALRN‐6924 potently inhibits the clonogenic capacity of megakaryocyte progenitors in MK‐CFU assays but it is less potent during the process of megakaryocyte maturation than the MDM2‐only inhibitor RG7388. These findings suggest that the disruption of later stages of megakaryocyte maturation more severely affects platelet production, ultimately resulting in thrombocytopenia. However, inhibition of less mature megakaryocyte progenitors, prior to entering terminal maturation, appears to be better tolerated. This may explain the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 vs. MDM2‐only inhibitors, and suggests that MDMX in bone marrow cells, where MDMX RNA expression is the highest of all 37 normal tissues reported in the Protein Atlas, may contribute to these differences.