PF-04449913 Reverts Multi Drug Resistance (MDR) by a Strong Down-Regulation of ABCA2 and BCL2 on Leukemia Stem Cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia Treated Patients
Authors: Papayannidis Cristina , Guadagnuolo Viviana , Iacobucci Ilaria , Durante Sandra , Terragna Carolina , Ottaviani Emanuela , Abbenante Maria Chiara , Cattina Federica , Soverini Simona , Lama Barbara , Toni Lucia , Levin Wendy J. , Courtney Rachel , Baldazzi Carmen , Curti Antonio , Baccarani Michele , Jamieson Catriona , Cortes Jorge E. , Oehler Vivian , McLachlan Karen , Van Arsdale Todd , Martinelli Giovanni 
Published: October 2019

Abstract 1429

The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. The Sonic Hedgehog (Hh) pathway is an essential regulator of multidrug resistance (MDR) in leukemia by exerting it's effect on leukaemia stem cells (LSC). PF-04449913 is a selective and potent small molecule inhibitor of the Hh pathway and leukemia self-renewal and is currently being evaluated in Phase I clinical trials.

In order to evaluate the activity of PF-0444913 in overcoming the drug resistance of leukemia stem cells, we studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies including Myelofibrosis (MF), MDS, Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic leukemia (CMML) and Acute Myeloid Leukemia (AML) patients. We were able to collect and separate highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) in 5 AML, 1 MF and 2 CML patients, by immunomagnetic separation, and analyze them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform.

We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Among these genes, we demonstrated a down regulation of Bcl2 (fold change −1.03004; p value= 0.01), ABCA2 (fold change −1.08966; p value=0.03), Bcl2l13 (fold change −1,04259; p-value=0,027642), Bcl2l2 (fold change −1,17214; p-value=0,000768), Casp4 (fold change −1,06551; p-value=0,032428), Casp7 (fold change −1,01569; p-value=0,006688), Casp10 (fold-change −1,3076; p-value=0,050431), ABCF1 (fold change −1,04999; p-value=0,07213).

On the contrary, ABCB1 (fold change 1,46592) and ABCG2 (fold change −1,16103) are respectively up and down regulated, with a not statistically significant p-value (0,35375 and 0,288194 respectively).

Bcl2 (B-cell lymphoma 2), Bcl2l2 (Bcl2-like protein 2) and Bcl2l13 (Bcl2-like 13) are the founding members of the Bcl-2 family of apoptosis regulator proteins. Recent studies showed that Hh signals upregulate Bcl2 to promote cellular survival.

Casp 4,7,10 (Caspases, or c ysteine-asp artic proteases) are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation.

ABCA2 (ATP-binding cassette sub-family A member 2), ABCF1 (ATP-binding cassette sub-family F member 1), ABCB1 (ATP-binding cassette sub-family B member 1, MDR1), ABCG2 (ATP-binding cassette sub-family G member 2) belong to the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. One mechanism of MDR is the increased expression of ABC drug transporters that mediate energy-dependent transport of drugs out of the cells against a concentration gradient, resulting in low intracellular drug concentrations. This is a common finding in LSC, and represents an important clinical problem for disease eradication.

Furthermore, we evaluated Gli1, Gli2 and Smo expression by GEP, comparing data before and after 28 days of treatment with PF-04449913 and, as expected, we observed a down regulation of Gli1 (fold change −1.0775), Smo (fold change −1.07702), and an up regulation of Gli2 (fold change 1.08191).

Our results suggest that PF-04449913 is able to revert MRD mechanisms of LSC by a strong down regulation of genes (Bcl-2, Bcl-2l13, Bcl-2l2, ABCA2, and ABCF1), which are critical for chemoresistance in acute and chronic leukemia patients. Therefore, the combination of PF-04449913 with Tyrosine Kinase inhibitors or conventional chemotherapy could represent a valid new therapeutic approach in these haematological malignancies.

Acknowledgments.

Work supported by Pfizer, European LeukemiaNet, FIRB 2008, PRIN 2009, AIRC, AIL, COFIN, University of Bologna and BolognAIL.

Disclosures:

Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jamieson:Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. McLachlan:Pfizer: Employment. Van Arsdale:Pfizer: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.