G. Vignir Helgason

Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells.

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

Kill one bird with two stones: potential efficacy of BCR-ABL and autophagy inhibition in CML

The introduction of imatinib in the treatment of chronic myeloid leukemia (CML) represents the most successful example of targeted therapy in human cancer. However, leukemic stem cells are insensitive to tyrosine kinase inhibitors (TKIs) and contribute to the persistence of disease by representing a reservoir of selfrenewing cells that replenish the disease after drug discontinuation. This finding has refocused the interest of scientists toward drug combinations, ie, treating with TKIs and simultaneously targeting alternative survival mechanisms. One candidate target mechanism is autophagy, a cellular recycling process that acts as a cytoprotective shield in CML cells in response to TKI-induced stress and in other cancer cells surviving in an inhospitable microenvironment. On that basis, inhibition of autophagy has now become an exciting option for combination treatment in cancer, and clinical trials have been initiated in solid and hemopoietic tumors such as CML, chronic lymphocytic leukemia, and multiple myeloma. This review describes the biology of CML and elucidates how the molecular driver BCR-ABL led to the development of TKIs. We then discuss the molecular regulation of autophagy and the potential for autophagy inhibition as the next step in our attempt to tackle the problem of CML persistence to offer a curative option.

The Antiproliferative Activity of Kinase Inhibitors in Chronic Myeloid Leukemia Cells Is Mediated by FOXO Transcription Factors

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR‐ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL‐specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR‐ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over‐expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA‐mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI‐induced apoptosis. These data demonstrate that TKI‐induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. Stem Cells 2014;32:2324–2337

Autophagy in blood cancers: biological role and therapeutic implications

Autophagy is a cell recycling process the molecular apparatus of which has been identified over the past decade. Autophagy allows cells to survive starvation and inhospitable conditions and plays a key role in numerous physiological functions, including hematopoiesis and immune responses. In hematologic malignancies, autophagy can either act as a chemo-resistance mechanism or have tumor suppressive functions, depending on the context. In addition, autophagy is involved in other important aspects of blood cancers as it promotes immune competence and anti-cancer immunity, and may even help enhance patient tolerance to standard treatments. Approaches exploiting autophagy, either to activate or inhibit it, could find broad application in hematologic malignancies and contribute to improved clinical outcomes. These aspects are discussed here together with a brief introduction to the molecular machinery of autophagy and to its role in blood cell physiology.

Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second- and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs). Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically. Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell–enriched (CD34+ and CD34+CD38−) and differentiated (CD34−) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope–assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease.

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

ABSTRACT A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Do we need more drugs for chronic myeloid leukemia

The introduction of protein tyrosine kinase inhibitors (TKIs) in 1998 transformed the management of chronic myeloid leukemia (CML), leading to significantly reduced mortality and improved 5 year survival rates. However, the CML community is faced with several clinical issues that need to be addressed. Ten to 15% of CML patients are diagnosed in advanced phase, and small numbers of chronic phase (CP) cases experience disease progression each year during treatment. For these patients, TKIs induce only transient responses and alternative treatment strategies are urgently required. Depending on choice of first line TKI, approximately 30% of CML CP cases show suboptimal responses, due to a combination of poor compliance, drug intolerance, and drug resistance, with approximately 50% of TKI‐resistance caused by kinase domain mutations and the remainder due to unknown mechanisms. Finally, the chance of successful treatment discontinuation is on the order of only 10–20% related to disease persistence. Disease persistence is a poorly understood phenomenon; all CML patients have functional Philadelphia positive (Ph+) stem and progenitor cells in their bone marrows and continue to express BCR‐ABL1 by DNA PCR, even when in very deep remission and following treatment discontinuation. What controls the maintenance of these persisting cells, whether it is necessary to fully eradicate the malignant clone to achieve cure, and how that might be approached therapeutically are open questions.

Mechanisms and novel approaches in overriding tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia is a stem cell-initiated but progenitor-driven disease induced by the BCR–ABL oncogene. Tyrosine kinase inhibitors (TKIs) were introduced in the late 1990s and have revolutionized the management of chronic myeloid leukemia in chronic phase. The majority of patients can now expect to live a normal life as long as they continue to comply with TKI treatment. However, in a significant proportion of cases TKI resistance develops over time, requiring a switch of therapy. The most frequent mechanism for drug resistance is the development of kinase domain mutations that reduce or completely ablate drug efficacy. Fortunately, the last 10 years have seen an impressive array of new drugs, some modeled on the mechanism of action of imatinib, others employing more novel approaches, for these patients.

AXL BLOCKADE BY BGB324 INHIBITS BCR-ABL TYROSINE KINASE INHIBITOR-SENSITIVE AND -RESISTANT CHRONIC MYELOID LEUKEMIA

Purpose: BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1–independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML. Experimental Design: We used primary cells from patients with CML and TKI-sensitive and -resistant BCR-ABL1+ CML cell lines and a novel ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small-molecule Axl inhibitor BGB324 in vitro and in vivo. In BCR-ABL1–unmutated cells, we also investigated BGB324 in combination with imatinib. Results: We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of patients with CML compared with healthy individuals and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34+ cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status. Conclusions: Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as -resistant CML and support the need for clinical trials. Clin Cancer Res; 23(9); 2289–300. ©2016 AACR.

Targeting BCR-ABL-Independent TKI resistance in chronic myeloid leukemia by mTOR and autophagy inhibition

Abstract Background Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4–6 mice per group). All statistical tests were two-sided. Results We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.