Dinner, Shira N.; Giles, Francis J.; Altman, Jessica K.
With improvements in supportive care and optimization of induction chemotherapy regimens, approximately 60% of adult patients with untreated acute myeloid leukemia (AML) will achieve a complete remission [1,2▪]. However, only 20% of all patients will have long-term disease-free survival. Although remission rates are often higher than 60% in younger patients, still only 40% of young patients that achieve a complete remission will have long-term disease-free survival, suggesting that the majority of patients will require salvage therapy for relapsed disease [1,2▪]. Screening for cytogenetic abnormalities and molecular abnormalities in nucleophosmin (NPM1), FMS-like tyrosine kinase 3 (FLT3), and CEBPA genes is now considered standard of care for prognostic evaluation at the time of diagnosis [3,4], and more recently high throughput sequencing technology identified validated mutations in DNA methyltransferase 3A (DNMT3A), TET2, and isocitrate dehydrogenases (IDH)1/2, which are known to impact prognosis [5–8,9▪▪]. Recently, whole-genome and exome sequencing of AML patient samples demonstrated, on average, only 13 gene mutations, lower than expected compared with other malignancies. The majority of samples had at least one mutation in one of nine categories of genes, including signaling genes, DNA-methylation-related genes, chromatin-modifying genes, the gene encoding nucleophosmin, myeloid transcription-factor genes, transcription-factor fusions, tumor-suppressor genes, cohesin-complex genes, and spliceosome-complex genes [10▪▪]. Many of these categories are of clinical translational relevance. The recognition of these mutations has not only impacted the ability to refine prognosis but also expanded the understanding of leukemogenesis, and is now leading to the development of targeted therapies for AML, which will be the focus of this review. We will highlight novel agents targeting epigenetic modulation, intracellular pathways, and the tumor microenvironment.
Epigenetic gene regulation is the control of transcription or gene expression through DNA methylation or histone modifications, without any changes to the underlying DNA sequence (Table 1) [11▪▪,12,13,14▪,15–17,18▪,19▪▪,20–24,25▪,26–29,30▪,31,32]. Epigenetic changes are thought to, at least partially, drive chemotherapy resistance in malignancies . Histone deacytlases (HDACs) are known to silence tumor suppressor genes and are a key therapeutic target of the HDAC inhibitors under investigation in AML, which include vorinostat, etinostat, and MGCD0103 [34▪,35▪,36]. Recent preclinical evidence highlights the importance of the IDHs, DOT1L, and DNMT3 in epigenetic modulation of AML and as potential therapeutic targets.
Mutations in the IDHs, IDH1 and IDH2, were recently identified in AML and impede oxidative decarboxylation of isocitrate to alpha-ketoglutarate . These mutations lead to novel enzymatic activity that results in production of R-2-hydroxyglutarate, an oncometabolite responsible for changes to DNA methylation, inhibition of histone lysine demethylases, block in cellular differentiation, and ultimately, tumorigenesis [11▪▪]. Prognostic significance of IDH mutations likely depends on the specific mutational locus and the presence of concurrent mutations of other genes, such as NPM1 and FLT3. The German–Austrian AML HD98A trial found that IDH mutations diminish the otherwise favorable prognosis seen in cytogenetically normal AML with mutated NPM1 and lack of FLT3-ITD mutation . The Acute Leukemia French Association 9801 and 9802 trials reported similar findings . However, Schnittger et al. observed that the unfavorable effect of IDH mutation was most obvious in AML with wild-type NPM1 status and in patients younger than 60 years of age. Green et al. reported no prognostic significance associated between IDH and NPM1 mutations, but when stratified by FLT3-ITD status, an IDH1 mutation was an independent adverse factor for relapse in FLT3-ITD(−) patients and a favorable factor in FLT3-ITD(+) patients.
Although further investigation is warranted in the clinical prognostic impact of IDH mutations, their role in tumorigenesis in human cancers, including glioma and AML, is clear and recent studies reported encouraging results with small molecule inhibitors of IDH [42▪,43▪▪]. AGI-6780 is a small molecule that inhibits the most commonly occurring IDH mutation in AML, IDH2-R140Q [43▪▪]. Treatment with this inhibitor lowered R-2-hydroxyglutarate to normal physiological levels in an erythroleukemia cell line and IDH2-mutated primary human AML cells. However, in the mutant primary human AML cells, a burst of proliferation occurred initially, followed by an increase in mature cell types at the expense of progenitor cells [43▪▪]. These results imply that mutant IDH2 inhibition can be used to promote differentiation of mutated AML cells. Based on preclinical results, a first in human study of the IDH2 inhibitor AG-221 is being conducted in patients with hematologic malignancies (NCT01915498).
MLL gene rearrangements at position 11q23 occur in approximately 10% of AML, acute lymphoid or mixed lineage leukemias and are associated with an aggressive disease course . Most translocations of the MLL gene result in oncogenic fusion proteins that interact with DOT1L, which is a histone methyltransferase enzyme that targets lysine 79 in the globular domain of histone H3 (H3K79). This leads to DOT1L hypermethylation and activation of MLL target genes that drive leukemogenesis [12,13,14▪]. Recently, a potent DOT1L inhibitor, EPZ-5676, was generated with a long half-life, and in vivo demonstrated 37 000-fold greater selectivity for DOT1L histone methyltransferase than any other methyltransferase tested [45▪▪]. In addition to inhibiting H3K79 methylation, MLL-fusion target gene expression was decreased. EPZ-5676 selectively killed acute leukemia cells with MLL translocations. In-vivo use of the small molecule in a rat model of MLL leukemia by continuous infusion produced complete tumor regression, which was sustained after discontinuing the agent [45▪▪]. Based on these findings, EPZ-5676 is in an early-phase clinical trial in patients with advanced hematologic malignancies, including acute leukemias harboring translocations of the MLL gene (NCT01684150).
DNA METHYLTRANSFERASE 3
High-throughput sequencing in AML recently identified mutations in the DNMT3A gene . In particular, mutations of the R882 codon impair methyltransferase activity . Although the exact impact of this finding on leukemogenesis remains unclear, it is thought that these mutations may influence response to treatment with azanucleoside DNMT inhibitors (’hypomethylating agents’) such as azacitadine or decitabine. In a study of elderly patients with untreated AML, decitabine treatment produced a complete remission of 47% and provided the greatest benefit in patients with low DNMT3A activity . Follow-up data from the same group reported DNMT3A-mutated patients had a higher median white blood cell count; DNMT3A mutations were significantly associated with NPM1 mutations, and a trend towards a higher prevalence of FLT3-ITD among DNMT3A-mutated patients. While the complete remission rate for the whole cohort was 41% (19/46), 75% (6/8) of DNMT3A-mutated patients achieved a complete remission . These data suggest that AML with mutations in DNMT3A is associated with increased response to azanucleoside DNMT inhibitors and warrants further investigation.
Inhibition of apoptosis by the overexpression of intracellular prosurvival proteins such as BCL-2 and RAF, as well as constitutive activation of and aberrant signaling through intracellular pathways such as the phosphoinosityl-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway and the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular-signal regulated kinase (ERK) pathway in the leukemia cells enhance survival, proliferation, and chemotherapy resistance  (Table 1) [11▪▪,12,13,14▪,15–17,18▪,19▪▪,20–24,25▪,26–29,30▪,31]. Thus, drugs that inhibit single or multiple proteins and pathways are actively under investigation.
The antiapoptotic protein BCL-2 is thought to drive leukemia stem cells and increase chemotherapy resistance in AML [17,18▪]. In a preclinical mouse model of high-risk myelodysplastic syndromes (MDS)/AML the small molecule inhibitor of BCL-2 homology domain 3 (BH3), ABT-737, down-regulated BCL-2, increased apoptosis, and extended the animal lifespan, suggesting effective targeting of the leukemia initiating cell [19▪▪]. When AML cells and xenograft AML models were exposed to both ABT-737 and a PI3-kinase inhibitor, expression of antiapoptotic proteins decreased and induced apoptosis along with tumor regression, suggesting a potential benefit for dual pathway inhibition [48▪,49▪]. Despite encouraging preclinical data for BCL-2 inhibition, prior clinical trials of the BCL-2 antisense oligonucleotide G3139 in acute leukemia were disappointing . ABT-737, the BH3 inhibitor, is currently under investigation in solid tumors only.
The PI3K/AKT/mTOR pathway drives cell cycling, replication, and death. PI3-K, a kinase near the cell surface, is activated by a number of receptor tyrosine kinases, including FLT3 . Constitutive activation of the PI3K/AKT/mTOR pathway occurs in 60–80% of cases of AML, most frequently in AML with a FLT3-ITD mutation, and is associated with shorter disease-free and overall survival [51,52]. In vitro, simultaneous blockade of PI3K with GDC-0941 and FLT3 with sorafenib in AML FLT3-ITD mutant cells inhibited prosurvival AKT signaling and promoted apoptosis [53▪]. As a single agent, another PI3K inhibitor, LY294002, induced apoptosis of AML cells in a dose-dependent manner [54,55]. Based on these data, BKM120, an oral PI3K inhibitor, is currently being evaluated in relapsed and refractory acute leukemias. (NCT01396499).
Several early-phase clinical trials investigated mTOR inhibitors in AML, such as rapamycin and deforolimus. These studies showed that the agents are effective in suppressing phosphorylation of downstream targets of mTOR but have limited clinical activity as monotherapy [21,22]. This may be because of the fact that these rapamycin analogues only target mTORC1, and do not inhibit mTORC2. OSI-027 targets both mTORC1 and mTORC2 and produced more potent antileukemic responses than selective mTORC1 targeting with rapamycin in AML cell lines . Results of a clinical trial with OSI-027 in solid tumors and lymphoma are awaited (NCT00698243). Another agent, NVP-BEZ235, inhibits mTORC1 and mTORC2, as well as PI3K, leading to a decreased proliferation rate and apoptosis in AML cells in vitro. BEZ235, is currently being evaluated in a clinical trial of relapsed or refractory acute leukemia (NCT01756118). Additional dual TORC and PI3K/mTOR inhibitors are currently in development.
Because of limited single-agent activity with rapalogues, preclinical studies have been conducted in combination with chemotherapy. When temsirolimus was combined with clofarabine, this led to increased apoptotic effects on AML cells . Modest clinical efficacy was demonstrated with this combination in elderly patients with relapsed and refractory AML. In 53 patients, 21% achieved a complete remission with a median disease-free survival of 3.5 months, and median overall survival of 4 months (9.1 months for responders) . Similarly, rapamycin added to mitoxantrone, etoposide, and cytarabine (MEC) in relapsed and refractory AML produced a 22% response rate . Another study evaluated the addition of everolimus to conventional daunorubicin plus cytarabine (’3 + 7’) in relapsed AML and demonstrated a response rate of 68% [25▪]. These data suggest further study is warranted of mTOR inhibitors or dual TORC inhibitors, given their increased antitumor activity, in combination with chemotherapy or other novel agents in AML.
Aberrant signaling through cell surface growth factor and cytokine receptors, RAS mutations, and RAF overexpression lead to constitutive activation of the MEK and ERK pathways. Deregulation of these pathways occurs frequently in AML, resulting in aggressive and refractory disease . AZD6244, an orally available inhibitor of the MEK kinase, decreased peripheral and marrow blasts, but failed to produce complete responses in patients with both newly diagnosed and relapsed AML in a phase II clinical trial . This may be explained by in-vitro data showing that MEK inhibition leads to compensatory upstream hyperactivation of RAF and/or parallel signaling through the PI3K/AKT/mTOR pathway, both of which may function as ‘escape’ pathways . In-vitro data demonstrated improved antitumor activity with dual inhibition of MEK and PI3K/AKT/mTOR pathways [60▪,61]. An upcoming trial will combine the oral MEK inhibitor, trametinib, with an oral AKT inhibitor, GSK2141795, in AML patients with RAS mutations (NCT01907815).
Stem cells within the bone marrow microenvironment are distinguished by their ability to proliferate, differentiate, and self-renew (Table 1). The cross-talk between the AML blast cells and the tumor microenvironment in the hematopoietic stem cell niche is thought to influence chemotherapy resistance and disease relapse . Therefore, targeting the interaction between blast cells and the microenvironment has been a focus of novel drug development.
Both normal stem cells and leukemic blasts express the chemokine receptor, CXCR4, which is essential to stem cell migration and engraftment in the bone marrow. Activation of the CXCR4 transmembrane receptor by its ligand, CXCL12, leads to increased signaling in the PI3K/AKT and MAPK pathways, ultimately causing cell proliferation and survival . Increased CXCR4 levels are associated with chemotherapy resistance and poor outcomes in AML [63▪▪,64]. Plerixafor is a small molecule antagonist of CXCR4 that mobilizes stem cells into the peripheral blood and sensitizes them to cytoxic chemotherapy . Based on these findings, a phase 1/2 study of plerixafor in combination with MEC chemotherapy was conducted in 52 patients with relapsed or refractory AML. Forty-six percent of patients achieved a complete remission and correlative studies demonstrated a two-fold mobilization in leukemic blasts into the peripheral circulation . Clinical trials investigating plerixafor in combination with decitabine and in combination with clofarabine are currently underway in older patients with AML (NCT01352650, NCT01160354).
BMS-936564/MDX-1338 is a fully human IgG(4) monoclonal antibody targeted against CXCR4. MDX-1388 demonstrated antileukemia activity in AML xenograft models and induced apoptosis in AML cell lines [67▪]. A phase I first in human study of MDX-1338 is currently underway in relapsed AML and select B cell hematologic malignancies (NCT01120457) After demonstrating safety in multiple myeloma, another high-affinity antagonist for CXCR4, BL-8040, is also currently being investigated in a phase IIa trial in relapsed and refractory AML (NCT01838395).
Provirus integration site for Moloney murine leukemia virus
Provirus integration site for Moloney murine leukemia virus (PIM) family proteins are serine/threonine kinases involved in oncogenesis. Three PIM kinases (PIM-1, 2, and 3) have been identified and regulate transcription, translation, cell cycle, survival, and drug resistance . In-vitro data suggest PIM-1 activity is essential for normal CXCR4 surface expression. PIM-1-deficient bone marrow resulted in ineffective cell migration and displayed decreased surface CXCR4 expression and impaired CXCL12–CXCR4 signaling. Increased PIM-1 produced high levels of CXCR4, which could be reduced with use of a PIM-1 inhibitor in vitro. These preclinical data suggest that PIM-1 inhibitors may interfere with leukemic blast cell interactions with the microenvironment leading to antitumor activity. Additional in-vitro and xenograft AML model data with the PIM-1 inhibitor SGI-1776 showed that PIM-1 inhibition induced apoptosis by blocking RNA and protein synthesis .
FLT3-ITD-mediated leukemogenesis has also been linked to CXCR4 through PIM serine/threonine kinases. FLT3-ITD-mutated leukemia is associated with increased expression of oncogenic PIM serine/threonine kinases . Another PIM-1 inhibitor, AR00459339, caused dephosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339, the PIM-1 inhibitor, with a FLT3 inhibitor resulted in additive cytotoxic effects in FLT3-ITD mutant AML cells. AR00459339 was cytotoxic to samples from patients with resistance to FLT3 inhibitors, suggesting a possible benefit to combining these agents in FLT3-ITD mutant AML . AZD1208 is an orally available, potent, and highly selective inhibitor that effectively inhibits all three PIM isoforms and has demonstrated activity in vitro and in xenograft models . A phase I trial of AZD1208 is underway in relapsed and refractory AML patients with either FLT3-ITD wild-type or mutated disease (NCT01489722). In this trial, it may be of particular interest to assess whether there is differential response between those patients with ITD mutated AML and those lacking the mutation.
Axl is a member of the Tyro3, Axl, Mer tyrosine kinase receptor family expressed on AML blast cells [30▪,72]. In vivo, AML cells stimulate bone marrow stomal cells in the microenvironment to express the Axl ligand growth arrest-specific gene 6 (Gas6), which drives cancer cell proliferation and survival [73,74]. Concurrent upregulation of Axl and Gas6 induces chemoresistance in AML cells and in-vivo data suggest AML cells have the ability to instruct the bone marrow stomal cells to increase Gas6 expression [30▪]. When Axl signaling is inhibited by a small-molecule Axl kinase inhibitor, BGB324, AML cell proliferation is reduced and apoptosis increases [30▪]. Additional in-vivo data demonstrated that phosphorylation of Axl resulted in FLT3 activation and Axl blockade inhibited the growth of FLT3-positive AML cells . This suggests that targeting Axl in both FLT3-ITD wild-type and mutated AML is a promising therapeutic strategy. The first in human clinical trial of BGB324 in AML will begin shortly.
Advances in AML treatment and survival outcomes have been limited in the last several decades. However, as molecular analysis of AML becomes more sophisticated and prevalent in clinical practice with further advances in next generation sequencing, treatment for AML is expected to evolve. The clinical impact of both individual and combined mutations will need to be discerned in order to direct selection of both conventional chemotherapy and novel targeted therapies. Decoding these mutations will continue to allow us to both improve prognosis and develop novel therapies for patients with AML. Extensive preclinical work has led to the identification of relevant pathways and the development of a large number of potential novel therapeutic agents. The pathways and compounds discussed in this review are of major clinical-translational interest and the results of the evolving clinical trials and continued laboratory advances are expected to lead the way to an even greater understanding of leukemogeneis and the development of more targeted therapies. The precise role of how to most effectively manipulate these mutations and pathways in combination with other novel agents or chemotherapy is still yet to be defined and expected to be the topic of future clinical trials.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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