The one-two punch: Combination treatment in chronic myeloid leukemia

Critical Reviews in Oncology/Hematology 88 (2013) 667–679

The one-two punch: Combination treatment in chronic myeloid leukemia Kendra L. Sweet, Lori A. Hazlehurst, Javier Pinilla-Ibarz ∗ Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, 13131 Magnolia Drive, 3 East, Rm 3056H, Tampa, FL 33612, United States Received 1 December 2012; received in revised form 31 May 2013; accepted 18 July 2013

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Are tyrosine kinase inhibitors curative for chronic myeloid leukemia? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Need for a one–two punch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The JAK-STAT pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. JAK2 in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. STAT3 in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. STAT5 in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. JAK/STAT pathway inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. JAK2 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. STAT3 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. STAT5 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hedgehog signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The Hh pathway in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Hh inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wnt/β-catenin pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The Wnt pathway in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Wnt pathway inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein phosphatase 2A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Role of PP2A in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. PP2A activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mTOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. mTOR complexes in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Dual mTORC2/mTORC1 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other drug classes/molecules with potential utility in CML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Interferon alfa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. HDACis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Alox5 (5-lipooxygenase) inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. HSP90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +1 813 745 1387; fax: +1 813 745 6817. E-mail address: [email protected] (J. Pinilla-Ibarz).

1040-8428/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.critrevonc.2013.07.017

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Abstract Despite the success of tyrosine kinase inhibitor (TKI) therapy in patients with chronic myeloid leukemia (CML), minimal residual disease persists, requiring indefinite treatment. Accumulated evidence has shown that leukemic stem cells (LSCs) in the bone marrow can survive TKI treatment via downstream BCR-ABL1–independent signaling pathways that are activated by soluble growth factors and interactions with the extracellular matrix in the bone marrow microenvironment. Research efforts have therefore turned to the identification and development of agents that target LSCs, and together with TKIs, have the potential to eradicate CML. A number of such agents are now under clinical investigation, and others are soon to enter early-phase studies. This review examines the pathways, molecular targets, and potential new therapeutics that, with TKIs, may provide an effective “one-two punch” to cure CML. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Imatinib; Nilotinib; Dasatinib; Chronic myeloid leukemia; Tyrosine kinase; Leukemic stem cells

1. Introduction 1.1. Are tyrosine kinase inhibitors curative for chronic myeloid leukemia? Chronic myeloid leukemia (CML) is caused by the constitutive activity of an aberrant tyrosine kinase (BCR-ABL1) that is created by the translocation of the Abelson (ABL) 1 oncogene on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22 [1–3]. The BCR-ABL1 kinase, in turn, drives the activation of a series of pro-survival signaling pathways that eventually lead to malignant transformation [4]. Understanding of the molecular basis of CML enabled the development of targeted treatment to block BCR-ABL1 activity, a strategy that proved highly successful. Initial phase III data from the international randomized study of interferon and STI571 (IRIS) demonstrated overwhelmingly positive results for imatinib, the first available BCR-ABL1 tyrosine kinase inhibitor (TKI), versus the standard treatment at that time, interferon plus cytarabine [5]. Indeed, imatinib completely altered the treatment paradigm, making long-term survival a new reality for most patients. Eight-year results of the IRIS study showed estimated overall survival (OS) to be 85%; OS was 93% if only CML-related mortality and deaths prior to stem cell transplant were considered [6]. Yet it was evident from IRIS that some patients experienced disease progression while on treatment with imatinib. Most events occurred early, within the first 3 years of treatment [6]. Further, the stop imatinib (STIM) study, which probed the possibility of imatinib discontinuation in patients who had achieved a complete molecular remission (CMR; defined as undetectable residual disease on quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR]) for at least 2 years, found that only select patients could cease treatment without relapse [7,8]. Even patients who do not relapse after discontinuation have evidence of disease when assessed by sophisticated methods such as qRT-PCR [9]. Indeed, some patients who are in prolonged remission with continued imatinib treatment may still harbor BCRABL1–positive stem cells [10]. Thus, despite its indisputable benefits, imatinib alone is unlikely to eradicate CML. Four more-potent TKIs, nilotinib, dasatinib, bosutinib, and ponatinib, were investigated and approved for the treatment of CML patients with intolerance of or resistance to

prior therapy including imatinib. Two of these agents, nilotinib and dasatinib, were evaluated in separate randomized, controlled studies as first-line treatment for CML in newly diagnosed patients. Nilotinib was compared with imatinib in the evaluating nilotinib efficacy and safety in clinical trials–newly diagnosed patients (ENESTnd) study [11], and dasatinib was compared with imatinib in the dasatinib versus imatinib study in treatment–naive cml patients (DASISION) [12]. Results of 4 years’ follow-up in ENESTnd and 3 years’ follow-up in DASISION are now available [13,14]. Nilotinib demonstrated a significantly higher rate of major molecular response (MMR) and CMR compared with imatinib, and dasatinib demonstrated a significantly higher MMR rate versus imatinib. The recent long-term data also show that fewer patients experienced disease progression with the newer agents; however, only the difference between nilotinib and imatinib was statistically significant [13,14]. Minimal residual disease remains evident with all available TKIs, however, making indefinite treatment necessary at this time. Although TKIs are generally well tolerated, long-term, potentially lifelong, treatment with TKIs increases the probability that patients will experience adverse events. The elimination of all residual disease would preclude the need for prolonged treatment and minimize the effect of TKI-related adverse events. It is clear that new strategies are needed in order to address these treatment gaps, with cure as the ultimate goal. 1.2. Need for a one–two punch In recent years, it has increasingly become appreciated that the bone marrow (BM) microenvironment provides a protective sanctuary in which leukemic stem cells (LSCs) can survive TKI treatment [15]. Normally, inhibition of BCR-ABL1 activity in CML cells leads to suppression of downstream pro-survival signaling pathways, tipping the balance toward pro-apoptotic mechanisms that lead to cell death. More-primitive progenitor cells that reside in the BM, however, are resistant to TKI-induced apoptosis, even though BCR-ABL1 activity is successfully inhibited [16–20]. It is believed that signaling pathways that are downstream of BCR-ABL1 remain intact in LSCs; these pathways may be activated by soluble growth factors and interactions with the extracellular matrix in the BM microenvironment, enabling

K.L. Sweet et al. / Critical Reviews in Oncology/Hematology 88 (2013) 667–679

LSC survival [15]. Therefore, recent research efforts have focused on the identification of suitable targets and the development of agents that can block BCR-ABL1-independent pro-survival pathways in LSCs. In this review, a number of key pathways and molecules implicated in the survival of LSCs are examined. In addition, we review agents that show promise for providing the necessary “second punch” that may, in combination with TKI therapy, be curative for CML. Many such agents are currently being tested in various solid tumors and other hematologic disorders; investigation of some of these agents for the treatment of CML has also begun.

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activation of downstream pathways, and ultimately interfered with oncogenic signaling [23]. 2.2. STAT3 in CML STAT3 is part of the signaling pathway downstream of JAK2 [23]. Data from in vitro CML models suggest a role for STAT3 in the development of TKI resistance in BM [24]. Bewry et al., used an in vitro coculture BM stroma model to determine the influence of BM soluble factors and extracellular matrices on imatinib sensitivity [24]. K562 cells (a transformed cell line derived from a CML patient), when cultured in BM stroma derived conditioned medium, were resistant to BCR-ABL1 inhibition. Drug resistance was associated with increased levels of STAT3. When STAT3 levels were reduced with small interfering RNA, K562 cells became sensitive to imatinib-induced cell death. Reducing STAT3 levels in regular growth conditions had no effect on imatinib sensitivity, suggesting that STAT3mediated drug resistance was due to factors present in the BM microenvironment. These data support a novel mechanism of BCR-ABL1-independent resistance and provide a rationale for the development of agents that can inhibit STAT3, thereby increasing the efficacy of TKIs in the BM.

2. The JAK-STAT pathway The janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is the principal signaling mechanism for a multitude of cytokines and growth factors [21]. JAK activation stimulates cell proliferation, differentiation, migration, and apoptosis, which are critical to numerous processes including hematopoiesis, immune system development, and mammary gland development (Fig. 1) [22]. 2.1. JAK2 in CML

2.3. STAT5 in CML In vitro and in vivo preclinical studies suggest that the JAK2 signaling pathway is activated in CML; inhibition of the JAK2 pathway reduced phosphorylation of BCR-ABL1 and

STAT5, like STAT3, is a downstream component of the JAK/STAT pathway [25]. In vitro studies demonstrated that

Cytokine ligand JAK2

JAK2

JA

P

K2

P

JA

Stat

Stat

P

K JA

2

St

P

St

at

at

St

P

K2

?

JA P

at

St

Ras P

at

P Stat

SOC3

Stat

K2

? Raf

PI3K SOC1 P

MEK

Akt

SOC1 SOC3

P mTOR

ERK

FoxO P

P ERK

Activation of genes important in proliferation and survival

Fig. 1. JAK-STAT pathway. Adapted with permission from Levine et al. [22].

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Table 1 Molecules in clinical development and their status in hematologic diseases. Molecule

Clinical development status for hematologic diseases

JAK2 inhibitors AZD1480

•Phase I and II study in PMF, PPV-MG, ET-MF [28]

CEP-701 (lestaurtinib)

•Phase I and II studies in MF [29,30] •Phase II study in FLT3 mutant AML [31] •Phase III study in infants with newly diagnosed ALL [32]

CYT387

•Phase I/II studies in PMF, PPV-MF, PET-MF [33,34]

LY2784544

•Phase I and II studies in MF, PV, and EF [35,36]

NS-018

•Phase I/II study in PMF, PPV-MF, or PET-MF [37]

Ruxolitinib (INCB018424)

•Indicated for intermediate or high-risk MF (PMF, PPV-MF, PET-MF] [38] •Phase I and II studies in PMF, PPV-MF and PET-MF [39–42] •Phase I and II study in refractory/relapsed AML or ALL [43] •Phase II study in AML, ALL, MDS, and CML [44,45] •Phase II study in relapsed/refractory DLBCL and PTCL [46]

SAR302503 (TG101348)

•Phase II and III studies in MF, PV-MF and ET-MF [47]

SB1518 (pacritinib)

•Phase I and II studies in AML, CML, CMML, MDS, MF [48,49] •Phase II studies in PMF, PPV-MF, PET-MF [50,51] •Phase I and II studies in HL, MCL, B-cell lymphoma [52,53]

STAT3 inhibitors ISIS 481464 (ISIS-STAT3Rx)

•Phase I study in advanced cancers [54]

OPB51602

•Phase I study in advanced hematologic malignancies (MM, NHL, AML, ALL, CML) [55]

Pyrimethamine

•Phase I/II study in CLL, SLL [56]

Hedgehog pathway agents BMS-833923 (XL139)

•Phase I study in multiple myeloma [57] •Phase II study in combination with dasatinib in CML [58]

GDC-0449 (vismodegib)

•Phase I study in multiple myeloma and plasma cell neoplasms [59]

IPI-926

•Phase II study in MF [60]

LDE225 (saridegib)

•Phase Ib study in combination with nilotinib in CML [61]

PF-04449913

•Phase I study in hematologic malignancies [62] •Phase Ib study in AML and MDS [63]

Wnt/␤-catenin pathway agents CWP232291

•Phase I study in acute myeloid leukemia [64]

PRI-724

•Phase I and II study in AML and CML [65]

Dual mTORC2/mTORC1 inhibitors CC-223

•Phase I/II study in advanced solid tumors, NHL and MM [66]

OSI-027

•Phase I study in patients with advanced solid tumors or lymphoma [67]

AML, acute myeloid leukemia; PMF, primary myelofibrosis; PPV-MF, post-polycythemia vera myelofibrosis; ET-MF, essential thrombocythemia myelofibrosis; PET-MF, post-essential thrombocythemia myelofibrosis; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; CML, chronic myelogenous leukemia; DLBL, diffuse large B-cell lymphoma; PTCL, peripheral T-cell lymphoma; CMML, chronic myelomonocytic leukemia; CLL, chronic lymphocytic leukemia; SLL, small lymphocytic leukemia; HL, Hodgkin lymphoma; MCL, mantle cell lymphoma; NHL, non-Hodgkin lymphoma; MM, multiple myeloma.

BCR-ABL1 can directly stimulate the tyrosine phosphorylation and dimerization of STAT5, bypassing JAK2, followed by the translocation of STAT5 dimers to the nucleus, where they promote activation of pro-survival genes [26]. More recently, high levels of STAT5 were shown to confer resistance to imatinib, nilotinib, and dasatinib, independently of JAK2. Whereas high levels of STAT5 were associated with reduced TKI-mediated cytotoxicity, reduction of STAT5 levels correlated with increased TKI-mediated killing. Further, STAT 5 levels from patient-derived cells correlated with TKI resistance and disease progression [27]. Although the authors

suggest that STAT5 inhibition may serve as an effective target for TKI resistance in patients with advanced disease [27], this strategy may also prove useful for eradicating CML cells residing in the protective BM microenvironment.

2.4. JAK/STAT pathway inhibitors The preclinical evidence to support the combination of JAK/STAT inhibitors with BCR-ABL TKIs is very promising.

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2.4.1. JAK2 inhibitors A number of small-molecule JAK2 inhibitors are in various stages of clinical development in hematologic diseases (Table 1), and one, ruxolitinib, has received US Food and Drug Administration (FDA) approval for the treatment of other myeloproliferative disorders [38]. A phase II study of ruxolitinib monotherapy in 38 patients with various refractory or relapsed hematologic malignancies, including two patients with CML, demonstrated that ruxolitinib provided modest activity as a single agent. Three patients, all with acute myeloid leukemia (AML) after having had a myeloproliferative disorder, showed a significant response [45]. Notably, samples from five patients, all of whom had elevated STAT3 levels at baseline, showed significantly decreased STAT3 levels 2 h after ruxolitinib dosing. Recently, a series of preclinical studies has provided a rationale for the clinical study of the combination of ruxolitinib and nilotinib in CML [68]. Conditioned medium from BM-derived mesenchymal stromal cells was shown to activate STAT3 in blastic-phase CML-derived cell lines and in LSCs obtained from patients with CML; STAT activation protected cells against nilotinib-mediated cell death. Ruxolitinib was able to block STAT3 activation and, when combined with nilotinib, sensitized both cell lines and patient-derived LSCs to the cytotoxic effects of nilotinib [68]. Utilizing small interfering–RNA strategies, Nair et al., demonstrated that reduced expression of JAK2 and TYK2 was required to sensitize cells to nilotinib. These results suggest that the combination of a promiscuous JAK inhibitor and BCR-ABL1 inhibition may be able to overcome the protective effects of the BM microenvironment and eliminate LSCs that are responsible for minimal residual disease. In our institution and based on our previous investigations, we have recently started a phase I/II trial to evaluate the toxicity and efficacy in achieving CMR of the combination of nilotinib and ruxolitinib. The future approval of other JAK-2 inhibitors and their availability in clinical practice will require the investigation of other combinations in the near future. 2.4.2. STAT3 inhibitors Several agents that specifically target STAT3 are in early clinical development, including one being evaluated in CML (Table 1). A phase I study of OPB-51602 is ongoing in Japan in patients with relapsed or refractory hematologic malignancies, including CML [55]. ISIS 481464, an antisense oligonucleotide inhibitor, is being examined in a phase I trial in patients with advanced cancers [54]. Pyrimethamine is an older agent used to treat malaria and other parasitic infections. It recently was shown to inhibit STAT3, and a phase I/II study in relapsed chronic lymphocytic leukemia/small lymphocytic leukemia has been initiated, with completion estimated for February 2014 [56].

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identified, by screening of compounds known to be safe in humans, to be a STAT5 inhibitor [69]. In vitro and ex vivo experiments demonstrated that pimozide decreases STAT5 target gene expression in KU812 and K562 CML cells and decreases colony-forming ability in patient-derived CD34+ BM cells from CML patients, but itself does not inhibit TK activity. Notably, the combination of pimozide with imatinib or nilotinib was synergistic for reducing STAT5 phosphorylation and decreasing the viability of KU812 cells, suggesting that such combinations may have clinical utility [69]. Indirubin is the major active antitumor component of a traditional Chinese herbal medicine that is reported to be useful in CML [70]. Preclinical studies have shown that compound E804, a potent derivative of indirubin, inhibits the STAT5 signaling pathway downstream of BCR-ABL1 and induces apoptosis of K562 CML cells, CML cells expressing the T315I mutation, and progenitor CD34+ CML cells from patient samples. 3. Hedgehog signaling pathway The Hedgehog (Hh) pathway, a signaling cascade that was initially identified in Drosophila melanogaster, is highly conserved among vertebrates [71]. It is essential for regulating the proliferation, migration, and differentiation of embryonic cells [72]. Aberrant reactivation of the pathway in adult tissue is associated with a wide variety of cancers, particularly basal cell carcinoma, medulloblastoma, and brain, gastrointestinal, breast, and prostate cancers. At a molecular level, in the absence of an Hh ligand, the patched (Ptch) receptor inhibits the action of smoothened (Smo), a G protein–coupled receptor. Binding of Hh ligands to Ptch relieves inhibition of Smo, which in turn activates zinc-finger like Gli proteins, leading to the transcription of tumor-promoting genes (Fig. 2) [71]. 3.1. The Hh pathway in CML Preclinical experiments have suggested that the Hh pathway is essential for the maintenance of LSCs in CML, and Smo, specifically, may be a rational therapeutic target. Smo is upregulated in LSCs relative to normal hematopoietic stem cells, and its inhibition in mice depletes LSCs, reduces the incidence of CML development, and, in combination with nilotinib, appeared to be more effective in preventing relapse than nilotinib alone [73]. In Smo knockout mice, hematopoietic stem cell renewal is impaired, and induction of CML by BCR-ABL1 is reduced [74]. A clinical strategy incorporating both Smo and BCR-ABL1 inhibition, therefore, may be successful in preventing the drug resistance and disease recurrence associated with TKI treatment alone [74]. 3.2. Hh inhibitors

2.4.3. STAT5 inhibitors The evaluation of agents that target STAT5 is at a preclinical stage. The psychotropic agent pimozide was recently

The preclinical data are convincing that the aberrant reactivation of the Hh pathway plays a significant role in

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OFF

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Fig. 2. Hedgehog signaling pathway. Adapted with permission from Mar et al. [71].

the preservation of LSCs. Among the Hh inhibitors currently being investigated for hematologic disorders (Table 1), two are being evaluated for CML. A phase Ib dose-finding study of the Smo antagonist LDE225 is being tested in combination with nilotinib in patients with CML in the chronic phase (CML-CP) who has failed prior treatment with other TKIs [61]. The other clinical trial is a large phase II randomized study evaluating the combination of the Smo inhibitor BMS833923 and dasatinib versus dasatinib alone in patients with CML-CP who did not achieve a major molecular response within 1 year of treatment with dasatinib alone. Some studies have shown that there are higher levels of Hh reactivation in the more advanced phases of CML than in CML-CP; if so, the combination of Hh or Smo inhibitors with TKIs may be more beneficial in advanced phases. The ongoing trials will better define the role of these compounds in different phases of CML.

displayed an enhanced capacity for self-renewal and leukemic potential [77]. The role of β-catenin in the maintenance of LSCs in CML was assessed in a mouse model [78]. Interestingly, deletion of β-catenin once CML was established did not increase survival in mice; however, β-catenin deletion plus imatinib synergized to eliminate CML stem cells that remained after imatinib treatment. This effect was also seen with pharmacologic inhibition of β-catenin with indomethacin [78]. Another research group has uncovered the involvement in CML of a little-known Wnt receptor, FZD-8, in a noncanonical Wnt/Ca2+ /nuclear factor of activated T-cell (NFAT) signaling pathway [79]. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production, and enhanced sensitivity to BCR-ABL1 inhibition in a mouse model. 4.2. Wnt pathway inhibitors

4. Wnt/β-catenin pathway The Wnt signaling pathway is a network of proteins involved in embryogenesis, in normal physiologic processes in adult animals, and in cancer [75]. Wnt signaling also appears to be a key pathway in stem cell regulation. In the canonical pathway, interaction of Wnt ligands with frizzled receptors and LDL receptor–related protein coreceptors blocks the degradation of β-catenin, an intracellular signaling molecule. The stabilized β-catenin then moves into the nucleus, where it induces the expression of target genes. In the case of mutations that block the degradation of β-catenin, target gene expression increases (Fig. 3) [76].

The demonstration that increased levels of β-catenin are associated with imatinib resistance and progression [77] makes use of a Wnt pathway inhibitor, or more specifically, a drug that directly inhibits β-catenin, a promising therapeutic avenue in combination with imatinib or other TKIs. Currently, there is one clinical trial investigating the safety and efficacy of Wnt pathway inhibition in patients with myeloid malignancies, including CML (Table 1). PRI-724, a specific inhibitor of cAMP response element-binding protein (CREB)-binding protein (CBP)/β-catenin, is being tested in a phase I/II dose-escalation study in patients with advanced malignancies. Patients with CML-CP who have failed two TKIs or patients with CML-AC/BC will be enrolled in the phase II portion of the study [65].

4.1. The Wnt pathway in CML Involvement of the Wnt pathway in CML was first described in 2004. Compared with normal stem cells from BM, progenitor CML cells from patients with accelerated phase/blast crisis (AP/BC) CML or patients who were resistant to imatinib demonstrated higher levels of βcatenin expression. Progenitor cells from these patients also

5. Protein phosphatase 2A Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that has been identified as an important tumor suppressor that negatively regulates many pro-survival signaling pathways. Inhibition of PP2A, specifically by the

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Fig. 3. Wnt signaling pathway. Adapted with permission from Trowbridge et al. [76].

SET oncoprotein, is associated with transformation in a range of human cancers [80,81]. 5.1. Role of PP2A in CML Perrotti et al. [82] have shown that BCR-ABL1–positive cell lines and CML progenitor cells derived from patients with CML-CP and -BC have enhanced expression of the PP2A inhibitor SET (Fig. 4). SET expression is higher in progenitors derived from patients with CML-BC than those with CML-CP, and higher expression is associated with progressive loss of PP2A-mediated tumor suppression [82,83]. Reactivation of PP2A has been shown to suppress growth, increase apoptosis, and decrease leukemogenesis in model systems; this effect may occur through the PP2A-mediated activation of a second tumor suppressor, Src homology region 1–domain phosphatase 1 (SHP-1), which can inactivate BCRABL1 [82,83].

6. mTOR pathway Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that forms the core of two multi-protein signaling complexes, mTORC1 and mTORC2, which regulate cell growth, protein synthesis, and cell-cycle progression

PP2A inhibition p210-BCR-ABL1 hnRNP A1

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FTY720

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Interestingly, preclinical research in CML has identified FTY720, a drug approved for the treatment of multiple sclerosis, as an activator of PP2A [82]. Presently, there are no clinical trials evaluating the combination of TKI and FTY720 in CML. In the future we may see trials combining this drug with TKIs in the setting of advanced phases of CML or TKI resistance.

SHP-1

A C B PP2A Impairment of leukemogenesis

Fig. 4. Protein phosphatase 2A (PP2A). Adapted with permission from Perrotti et al. [82].

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Fig. 5. mTORC1 and mTORC2 signaling complexes. Adapted with permission from Foster and Fingar [85].

via complex interactions with numerous signaling pathways, including phosphatidylinositol 3-Kinase/Akt [84]. mTORC1 and mTORC2 are distinguished by their sensitivities to the mTOR inhibitor rapamycin and by their distinct partner proteins, substrates, and cellular functions. mTORC1, which is formed by mTOR, Raptor and mLST8, is allosterically inhibited by rapamycin and other analogs of rapamycin (e.g., temsirolimus, everolimus). mTORC2, which includes mTOR, Rictor, mLST8, and SIN1, is not inhibited by rapamycin and its analogs (Fig. 5) [85].

6.2. Dual mTORC2/mTORC1 inhibitors In vitro data show that the dual mTORC inhibitors induce apoptosis in both mutated and unmutated BCR-ABL–expressing cells [86,88]. Two dual mTORC2/mTORC1 inhibitors are in early clinical development (Table 1): OSI-027 and CC-223. Neither of these trials includes patients with CML. However, the combination of dual mTORC inhibitors with TKIs has good potential to induce apoptosis in LSCs that are resistant to TKIs.

A. 6.1. mTOR complexes in CML Recent preclinical research has demonstrated that rapamycin-insensitive (RI)–mTORC1 complexes are activated in BCR-ABL-expressing cells and are involved in the translation of genes that mediate mitogenic responses. mTORC2 complexes are also activated in, and are important for the growth and survival of, BCR-ABL1–expressing cells [86]. The mTORC1 inhibitor everolimus has shown promise in a phase I/II clinical trial of patients with relapsed or refractory hematologic malignancies; however, CML patients were not included in the trial [87]. Notably, a dual mTORC2/mTORC1 inhibitor, OSI-027, potently suppressed LSCs from CML patients, providing a rationale for incorporating such agents in combination therapy for CML (Fig. 6) [86].

B. LSCQ LSCP

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LSCP

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EPC

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LPC

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LC

Mobilize quiescent LSCs: • HDACinhibion • HSP90 inhibion • Interferon alfa • mTORC inhibion Target self renewal pathways: • Alox5 inhibion • Hh/Smo inhibion • JAK2 inhibion • PP2A acvaon • Wnt/ catenin inhibion

Fig. 6. Approaches to the eradication of leukemic stem cells (LSCs). A. LSCs cycle between quiescent (LSCQ ) and proliferative (LSCP ) states. LSCP have the capacity for self-renewal. Eventually, LSCP differentiate into early progenitor cells (EPC), which become late progenitor cells (LPC) that mature into leukemic cells (LC). B. Two main approaches to eradication of LSCs: mobilization of LSCQ and targeting of self-renewal pathways.

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7. Other drug classes/molecules with potential utility in CML

ongoing phase I study (NCT00686218) is investigating the combination of imatinib and panobinostat.

A number of other molecules and drug classes are also being evaluated in hematologic disorders, including interferon alfa, histone deacetylase inhibitors (HDACis), an Alox5 inhibitor, and heat shock protein (HSP) 90 inhibitors.

7.3. Alox5 (5-lipooxygenase) inhibitor

7.1. Interferon alfa Interferon alfa has been an important treatment modality for CML for decades, although it has not been considered standard of care for CML-CP since the introduction of TKIs. Interest in interferon alfa for CML has shifted focus since observations were made that prior interferon alfa therapy may be correlated with sustained treatment-free remission after discontinuation of TKI therapy in clinical studies [89,90]. These clinical observations, in light of preclinical findings that exposure to interferon alfa can activate quiescent hematopoietic stem cells in a mouse model [91], suggest that prior or concomitant treatment of patients with interferon alfa can sensitize LSCs to eradication by TKI therapy. Several clinical studies have examined the combination of interferon alfa plus imatinib in patients with CML-CP [92–95]. Overall, the combination improves response rates or induces more-rapid responses over standard-dose imatinib alone, but the combination also increases the incidence of treatment-related adverse events. At present, others studies are ongoing that will evaluate the combination of interferon alfa plus nilotinib or imatinib (NCT00573378) and interferon alfa plus dasatinib (NCT01725204). Whether the combination of interferon alfa plus imatinib or other TKI results in LSC elimination remains to be determined and future trials with exhaustive correlative studies may answer this question. 7.2. HDACis Small-molecule HDACis have shown significant biological effects in preclinical models of oncogenesis; they induce growth arrest, differentiation, and apoptosis in vitro and in vivo, and can kill both actively proliferating and nonproliferating cancer cells [96,97]. In experimental models of CML, cotreatment with imatinib and the pan-HDACi LBH-589 induced apoptosis in nonproliferating, otherwise imatinib-resistant LSCs, significantly reduced LSC capacity to repopulate marrows of immunodeficient mice, reduced the number of putative CML LSCs in a transgenic mouse model, and reduced expression of genes related to primitive stem cell state [98]. Many such inhibitors are now in clinical trials, with several early-phase studies examining the combination of a TKI and HDACi in patients with CML. One phase I study (NCT00816283) has examined dasatinib in combination with vorinostat (no results have been published) and one

In a preclinical model of CML, Chen and colleagues have identified the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator of LSCs [99]. BCR-ABL1 does not induce CML in BCR-ABL1-transduced Alox5-/-mice, and Alox-deficient mice lack LSCs. These findings led to a phase I study of zileuton, a specific 5-lipoxygenase inhibitor approved for the treatment of asthma, in patients with CMLCP already receiving imatinib (NCT01130688). The study has a planned completion date of December 2014. 7.4. HSP90 inhibitors HSP90 is a molecular chaperone protein necessary for cellular survival [100]. HSPs monitor protein folding, prevent misfolding, and restore three-dimensional protein structure, activities that are essential during toxic cellular stress. Oncogenic cells depend on their ability to withstand endogenous (anoxia, nutrient deprivation, pH changes, and deranged signaling pathways) and exogenous (chemotherapy and radiation therapy) stressors for survival; inhibition of HSP90 in multiple cancer animal models has shown significant antitumor effects. Although the results of early efficacy studies of HSP90 inhibitors were encouraging, the ideal use of HSP90targeted therapeutics will require a deeper understanding of the functional differences in malignant neoplastic disease and normal cellular physiology.

8. Summary The BCR-ABL1 TKIs have been a groundbreaking success in treating CML. However, these agents may be unable to completely eradicate CML. Although long-term treatmentfree periods have been noted in some patients who have discontinued TKI treatment, evidence of minimal residual disease remains. Experimental studies suggest that quiescent LSCs survive TKI treatment because of microenvironmental factors in the BM. Novel pathways and molecules have been the focal point of drug development to target LSCs. It is anticipated that agents that inhibit critical pathways in LSCs, in combination with TKIs, may provide the one-two punch that is needed to eradicate CML.

Conflict of interest statements Javier Pinilla-Ibarz has received honoraria and research support from Bristol-Myers Squibb and Novartis. Kendra L. Sweet and Lori A. Hazlehurst have no conflicts of interest to report.

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Reviewers Elias Jabbour, M.D., Associate Professor, University of Texas MD Anderson, Leukemia, 1515 Holcombe Blvd, Department of Leukemia, Unit 428, Houston, TX 77030, United States. Photis Beris, Associate Professor, Faculté de Médecine, Geneva University, Swiss Internal Medicine, 51 avenue Blanc, CH-1202 Genève, Switzerland. Michael J. Mauro, M.D., Professor, Oregon Health Science University, Division of Hematology and Medical Oncology, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098, United States. Francis Giles, M.D., Professor, NUI Galway, HRB Clinical Research Facility, Geata an Eolais, University Road, Galway, Ireland. Dominik Heim, Head of Hematology Department, University Hospital, Basel, Spitalstrasse 21, Petersgraben 4, CH-4031 Basel, Switzerland.

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Acknowledgements Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals Corporation. We thank Mariana Ovnic, PhD, and Anna Lau, PhD, of Percolation Communications LLC for medical editorial assistance.

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Biographies Kendra L. Sweet, MD: Dr. Kendra Sweet is Clinical Instructor in Hematology and Medical Oncology at the H. Lee Moffitt Cancer Center and the University of South Florida in Tampa, Florida. Dr. Sweet is currently involved in a clinical

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trial looking at combination therapy in CML and has done research on prognostic factors and outcomes in CML. She has presented her findings at annual meetings of the American Society of Clinical Oncology, the American Society of Hematology, and the European School of Hematology. Lori A. Hazlehurst, PhD: Dr. Hazlehurst is an associate member of the Molecular Oncology program and a member of the Malignant Hematology program at the H. Lee Moffitt Cancer Center in Tampa, Florida. Dr. Hazlehurst’s research is focused on the identification and targeting of mechanisms of drug resistance emanating from the bone marrow microenvironment. Her work has been published in journals including Blood, Cancer Research, Molecular Cancer Therapeutics, and Leukemia Research. Javier Pinilla-Ibarz, MD, PhD: Dr. Javier Pinilla-Ibarz is an associate member of the Malignant Hematology and Immunology program at the H. Lee Moffitt Cancer Center in Tampa, Florida, and is an Associate Professor in the Department of Oncologic Sciences, College of Medicine, University of South Florida, Tampa, Florida. Dr. Pinilla-Ibarz is leading the chronic leukemia program at Moffitt. The program’s principal clinical interest is the treatment of patients with myeloid and lymphoid chronic leukemia. In this capacity, Dr. Pinilla-Ibarz has participated in multiple clinical trials involving tyrosine kinase inhibitors (TKIs). Dr. Pinilla-Ibarz is also the Director of Immunotherapy for Malignant Hematology. He has been involved in developing new immunotherapeutic approaches to the treatment of chronic myeloid leukemia. He is currently leading several clinical trials of peptide and cellular vaccines in different hematologic disorders. Other research areas include the role of the bone marrow microenvironment in mediating resistance to TKIs and the interaction of TKIs with the immune system. Dr. Pinilla-Ibarz frequently presents his findings at annual meetings of the American Hematology Association and the American Society of Clinical Oncology, and other national/international meetings. His work has been published in journals including Blood, Nature Immunology, Seminars of Hematology, and Leukemia. He has also authored multiple book chapters.