Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML
Authors: Helgason G.Vignir , Allan Elaine K , Mariani Samanta , Mukhopadhyay Arunima , Karvela Maria , Galavotti Sara , Audia Alessandra , Hamilton Ashley , Nicolini Franck E. , Druker Brian J. , Salomoni Paolo , Calabretta Bruno , Holyoake Tessa L. 
Published: October 2019

Abstract
Abstract 1664
Imatinib (IM), a tyrosine kinase inhibitor (TKI), represents the most successful targeted therapy in human cancer. However, this success has been tempered by problems of disease persistence and resistance. We have shown that disease persistence arises from a population of CML stem cells that survive despite complete inhibition of Bcr-Abl by TKI and that in CML stem cells autophagy is induced in response to TKI. Of critical importance, IM-induced autophagy provides a survival mechanism as autophagy inhibition using chloroquine combined with TKIs resulted in almost complete elimination of CML stem cells (Bellodi JCI 2009; Helgason Blood 2011). This pre-clinical work has now progressed to CHOICES clinical trial in which CML patients with residual disease on IM are randomised to continue IM or to have hydroxychloroquine (HCQ) added for 12 months (NCT01227135).
In addition to disease persistence, TKI resistance represents a major clinical challenge and can be caused by mutation in Bcr-Abl affecting drug binding (Bcr-Abl dependent) or by activation of alternative signalling pathways (Bcr-Abl independent). As none of the currently licensed TKIs inhibit Bcr-Abl carrying the T315I mutation we aimed to use ponatinib, a 3rd generation TKI, to tackle Bcr-Abl dependent resistance. For cells that have acquired Bcr-Abl independent mechanisms of resistance we aimed to test drugs that target alternative survival pathways and in both models to examine if they induce cell death in TKI-resistant cells, induce protective autophagy and synergise with autophagy inhibition to enhance elimination of CML stem/progenitor cells.
We demonstrated that ponatinib was effective in inhibiting proliferation and inducing apoptosis in chronic phase (CP) CML CD34+ cells (n=5) and in CP cells carrying the T315I mutation (n=2) in a dose-dependent manner. In addition, ponatinib, like 1st and 2nd generation TKIs, induced autophagy in CP CD34+ and T315I expressing cells. HCQ-mediated autophagy inhibition enhanced the effect of ponatinib in CML stem/progenitor cells with reduction in Colony Formation Cell (CFC) number increased from 34% with 100nM ponatinib alone to 77% in ponatinib/HCQ treated cells (n=4). This combination had no significant effect on proliferation or CFC formation of normal CD34+ cells (n=3). Most strikingly, ponatinib/HCQ combination resulted in near complete elimination of CML stem cells with less that 1% surviving treatment in Long-Term Culture Initiating Cell (LTC-IC) assay (n=3).
The PI3K/Akt/mTOR pathway, downstream of Bcr-Abl, provides an alternative drug target for CML patients with Bcr-Abl independent TKI-resistance. Treatment with clinically relevant concentrations of the dual PI3K/mTOR inhibitor BEZ235 resulted in dose-dependent induction of apoptosis in CP CML CD34+ cells (n=5) and in ponatinib-resistant KCL22 cells (generated by culturing cells in increasing concentrations of ponatinib for a prolonged period), whereas rapamycin, a less potent mTOR inhibitor, exhibited only anti-proliferative effects and failed to induce apoptosis. We also showed that inhibition of mTOR leads to induction of autophagy. Furthermore, autophagy inhibition, by Atg7 knockdown or HCQ treatment, augmented death induced by PI3K/mTOR inhibitors in CML cells, including ponatinib-resistant KCL22 cells. In CP CD34+ cells 100nM BEZ235 induced 12% apoptosis above untreated controls (n=6) and 65% reduction in CFC (n=8), that was increased to 18% and 83%, respectively, when combined with HCQ. When combined with HCQ-mediated autophagy inhibition, BEZ235 also reduced CFC potential of progenitor cells derived from 2 patients who have failed to achieve CCyR following any generation TKI, by 75% compared to 42% following ponatinib treatment. BEZ235 as monotherapy reduced survival of CML stem cells by 59% with the effect further increased in combination with HCQ, resulting in 74% reduction in stem cell number, indicating that autophagy also protects CML stem cells from death induced by PI3K/mTOR inhibition.
Taken together these data indicate that autophagy inhibition might not only potentiate treatment for CML patients responsive to TKI treatment by enhancing elimination of CML stem cells, but may represent an improved treatment option for both Bcr-Abl dependent and independent mechanisms of TKI-resistance in CML patients.

Acknowledgments:
Grants from KKLF (KKL404) and MRC (G0900882, CHOICES, ISCRTN No. 61568166)

Disclosures:
Nicolini: Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Druker:MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other.