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670 | CANCER DISCOVERY july 2017 www.aacrjournals.org

IN THE SPOTLIGHT

novel Mitochondrial Mechanisms of Cytarabine Resistance in Primary AML Cells Aaron D. Schimmer

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada . Corresponding Author: Aaron D. Schimmer , Princess Margaret Cancer Centre, Room 7-417, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada. Phone: 416-946-2838; Fax: 416-946-6546; E-mail: aaron.schimmer@uhn.ca doi: 10.1158/2159-8290.CD-17-0476 ©2017 American Association for Cancer Research.

summary: Farge and colleagues describe a novel in vivo approach to identify and study primary acute myeloid leukemia (AML) cells that persist in the marrow after chemotherapy. They discovered that AML cells that persist in the mouse marrow after treatment with cytarabine have increased oxidative phosphorylation and that inhibit-ing oxidative phosphorylation can restore sensitivity to cytarabine. Cancer Discov; 7(7); 670–2. ©2017 AACR.

See related article by Farge et al., p. 716 (8).

Acute myeloid leukemia (AML) is an aggressive hemato­logic malignancy characterized by the clonal expansion of cells that are blocked in their capacity for normal differentia­tion. Through biological and genetic studies, the molecular origins of leukemia and the mutations associated with this disease are being identifi ed. For example, driver mutations in genes such as NPM1 and IDH1/2 have been discovered in subsets of patients ( 1, 2 ), whereas other mutations, such as DNMT3A , have been reported in preleukemic cells and can predispose some patients to develop AML ( 3 ). Adding to the heterogeneity of this disease, AML is arranged in a cellular hierarchy. Rare stem and progenitor cells give rise to more abundant committed cells. Thus, effective therapies for AML must be able to target these genetic subclones and different cellular fractions.

The dramatic advances in understanding the biology of AML are now being translated into more effective thera­pies, and these new therapies are desperately needed, as classic chemotherapy is largely unchanged over the past 30 years. Traditionally, patients with newly diagnosed AML are treated with induction and consolidation chemotherapy consisting of the nucleoside analogue cytarabine and an anthracycline such as daunorubicin or idarubicin. Recently, the addition of the FLT3 inhibitor midostaurin to standard induction and postremission therapy was shown to decrease the risk of relapse and improve overall survival in patients with FLT3 mutations ( 4 ). In addition, the liposomal encap­sulation of cytarabine and daunorubicin in fi xed molar ratio (CPX­351) improved rates of remission and survival for patients with secondary AML or AML with high­risk cytoge­netics ( 5 ). Yet, for most patients, outcomes still remain poor due to primary refractory or relapsed disease. For example, in unselected patients under age 60 receiving standard

induction chemotherapy with cytarabine and daunorubicin, approximately 40% do not achieve remission with their fi rst cycle of therapy ( 6 ). In high­risk patients, such as older indi­viduals with high­risk cytogenetics and/or secondary AML, approximately 70% do not achieve remission. Moreover, for patients who achieve remission, relapse rates are fre­quently high, and in patients under 60, survival at 2 years is approximately 40%. In high­risk patients, such as those with AML secondary to myeloproliferative neoplasms, almost all relapse without transplant. As such, primary refractory and relapsed AML continue to represent large unmet needs in this disease.

Thus, in addition to understanding the biological basis for AML, it is equally important to understand the mecha­nisms by which AML cells are or become resistant to chemotherapeutic agents, including cytarabine and dau­norubicin. Understanding the mechanisms of resistance may lead to new combination therapies to overcome resist­ance or identifi cation of patients who should be treated with alternate regimens. To date, both cell­autonomous and cell­nonautonomous mechanisms of chemoresistance have been identifi ed. For example, microenvironment fac­tors, such as the overexpression of chemokines like SDF1 (stromal cell–derived factor 1) by marrow stromal cells or increases in marrow adipocytes, can render AML cells resist­ant to chemotherapy in vitro and in vivo . Likewise, increased expression of drug effl ux pumps such as p­glycoprotein, antiapoptotic proteins such as BCL2, or enzymes that metabolize cytarabine such as SAMHD1 are cell­dependent mechanisms of cytarabine resistance. To date, however, tar­geting resistance mechanisms has not been proven effective in phase III clinical trials, in part because these approaches also increase the sensitivity of normal cells to chemothera­peutic agents.

A limitation of many reports examining mechanisms of chemoresistance is that the experiments were restricted to studies of cell lines treated in culture. Fewer studies have examined chemoresistance in primary patient samples and rarely has chemoresistance in primary samples been evalu­ated in vivo . As such, identifying mechanisms of chemo­resistance that are relevant in the clinical setting has been challenging. A recent exception has been the demonstration that leukemic stem cells contribute to chemoresistance in

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july 2017 CANCER DISCOVERY | 671

patients with AML. In a new study by Ng and colleagues (7), patients with AML were scored for stemness based on a gene expression signature of leukemia stem cells. In multiple patient cohorts, a high degree of stemness, as measured by an increased LSC17 score, predicted for decreased rates of remis­sion and increased rates of relapse.

In this issue of Cancer Discovery, Farge and colleagues report a new model to evaluate mechanisms of chemo­resistance of primary cells in vivo and have discovered a new mechanism of drug resistance related to mitochondrial metabolism (8). In a series of elegant experiments, Farge and colleagues utilized a mouse model to evaluate AML cells that survive chemotherapy. In their model, immunodeficient NSG mice were engrafted with primary AML cells and then treated with cytarabine at a dose and schedule (60 mg/kg/day for 5 days) to reduce the level of leukemia in the periph­eral blood below detectable levels (Fig. 1). On day 8, the time of maximal depletion of AML cells in the blood, the AML cells that persisted in the mouse marrow were isolated and studied. Despite all samples having undetectable levels of leukemia in the blood, the reduction of cells in the marrow was variable and ranged from 4­ to 46­fold. The surviving fraction of cells retained their resistance to cytarabine when isolated and studied in vitro. These cells were also cross­resistant to other chemotherapeutic agents, including idaru­bicin and etoposide. The authors searched for mechanisms of resistance. Chemoresistance could not be explained by the persistence of leukemic stem cells, as only 3 of 22 tested cases were enriched for leukemic stem cells in the persisters. Rather, Farge and colleagues showed that the persisting cells from the majority of tested patients had different mitochon­drial and metabolic characteristics compared with the bulk population of AML obtained from control­treated mice. The persisting cells had increased mitochondrial mass, mito­chondrial membrane potential, ROS production, and a gene signature associated with oxidative phosphorylation, thus potentially representing a population of cells with greater rates of oxidative phosphorylation. Importantly, the authors demonstrated that reducing oxidative phosphorylation by inhibiting mitochondrial processes, such as fatty­acid oxida­tion, respiratory chain complex activity, mitochondrial DNA replication, or mitochondrial protein synthesis, increased the sensitivity of these resistant cells to cytarabine both in vitro and in vivo.

Thus, the work by Farge and colleagues represents a novel approach to evaluating chemoresistance in primary AML samples. By focusing on cells that persist in the mouse after maximal treatment, future studies can test whether new therapeutic strategies, even unrelated to metabolism, can eradicate the cells that persist after treatment with cytara­bine. For example, in samples with FLT3 or IDH mutations, would the addition of FLT3 or IDH inhibitors to cytarabine change the abundance of residual cells? In addition, would the persisting cells have the same mechanisms of resistance related to oxidative phosphorylation, or would new mecha­nisms emerge?

Farge and colleagues also suggest that this model might identify clinically relevant mechanisms of drug resistance. Their mouse model could be a surrogate for primary refrac­tory disease, as it captures cells that persist following a single cycle of chemotherapy and therefore could mimic the patient that clears the vast majority of bulk blasts but has regrowth of disease within a month of starting therapy. The model could also shed light on mechanisms of relapse. Although the high oxidative phosphorylation gene signature did not cor­relate with the probability of achieving remission with induc­tion chemotherapy in an independent dataset of patients with AML, it did correlate with overall survival, supporting the clinical relevance of their finding.

The work also opens new fields for investigation and future study. It is not fully understood how sensitivity to cytara­bine would be influenced by the metabolic state of the cell. Cytarabine is a structural analogue of deoxycytidine that is phosphorylated in cells to the active triphosphate form. The active metabolite is an inhibitor of DNA polymerase and is incorporated into DNA where it causes chain termination, thus blocking in DNA synthesis. However, it remains to be determined why increases in mitochondrial metabolism and oxidative phosphorylation would render cells resistant to this drug and the other tested cytotoxics.

Nonetheless, the finding that the persisting AML cells have increased oxidative phosphorylation builds on prior studies demonstrating that AML cells are unique in their mitochon­drial characteristics with a heightened reliance on oxidative phosphorylation (9) and decreased spare reserve capacity (10). Moreover, these results provided further evidence to support testing therapies that target mitochondrial metabo­lism and oxidative phosphorylation in AML.

Figure 1.  A novel in vivo approach to identify mechanisms of chemoresistance in AML. Primary AML cells were injected into immunodeficient NSG mice. After engraft-ment, mice were treated with cytarabine to reduce the leukemia in the peripheral blood below the level of detection. On day 8, at the time of maximal depletion of leukemia in the blood, the leukemic cells persisting in the mouse femur were isolated and studied. Persisting cells had increased oxidative phosphorylation as evidenced by increased mitochondrial mass, mitochondrial membrane potential, reactive oxygen species (ROS), and an oxidative phosphorylation OXPHOS gene signature.

Mitochondrial massMitochondrial membrane potentialROSOXPHOS gene signature

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672 | CANCER DISCOVERY july 2017 www.aacrjournals.org

Disclosure of Potential Conflicts of InterestA.D. Schimmer has received honoraria from the speakers bureau

of Novartis and is a consultant/advisory board member for the same.

Published online July 6, 2017.

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