Tavor et al. [21] reported the importance of the CXCL12-CXCR4 axis in the regulation of in vivo motility and development of human AML stem cells and identified CXCR4 neutralization as a potential treatment for AML. They found that all AML cells tested expressed internal CXCR4 and CXCL12, even cells without surface CXCR4 expression, and observed an antileukemia effect of the in vivo CXCR4 neutralization by blocking antibody in an AML xenograft model. Importantly, CXCR4 inhibition did not significantly affect the engraftment of normal human progenitors into nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Subsequently, several groups explored whether the US Food and Drug Administration (FDA)-approved small molecular CXCR4 inhibitor, plerixafor (AML3100), affected the trafficking and survival of AML cells in vitro and in vivo. AMD3100 caused inhibition of the CXCL12-induced migration of AML blasts, an outgrowth of leukemia colony forming units, and a promigratory effect of CXCL12 on AML engraftment into NOD/SCID mice [22]. Another study also demonstrated that AMD3100 inhibited proliferation in AML cell lines and triggered changes that mimicked differentiation [23].

In 2009, two preclinical studies evaluated the efficacy of two small molecular CXCR4 inhibitors, AMD3100 and AMD3465, in combination with chemotherapy or targeted agents, in human AML xenograft models. These studies demonstrated that these CXCR4 inhibitors exerted chemosensitizing effects, which supported the notion that the CXCL12-CXCR4 axis plays a key role in chemoresistance via leukemia-stromal interactions [4,5]. Zeng et al. [5] reported that CXCR4 inhibition by AMD3465, a derivative of AMD3100, increased the sensitivity of FLT3 inhibitors sorafenib and cytarabine in xenograft models of mouse leukemic cells with or without an FLT3-mutation. AMD3465, alone or in combination with granulocyte colony-stimulating factors (G-CSF), induced the mobilization of AML cells and progenitor cells into the circulation and enhanced the antileukemia effects of cytarabine and sorafenib, resulting in a reduced leukemia burden and prolonged survival in mice. Further, CXCR4 inhibition antagonized the stroma or CXCL12-induced chemotaxis and CXCL12-induced activation of prosurvival signaling pathways, such as PI3K/AKT and MAPK/ERK, in leukemic cells. In a separate report, Nervi et al. [4] showed the chemosensitizing effect of AMD3100 in acute promyelocyte leukemia (APL) mouse models with cytarabine, and the administration of AMD3100 to leukemic mice induced an increase of leukemic cells in blood, which peaked at 3 hours and returned to baseline by 12 hours.

Chen et al. [12] exploited the novel mechanism of chemosensitization in AML, and identified a CXCR4/let-7a/BCL-XL axis that may be responsible for the chemoresistance. They found that the overexpression of surface CXCR4 and activation of CXCR4 signaling results in downregulation of microRNA let-7a, which led to increased expression of the antiapoptotic protein BCL-XL in AML cells, suggesting that let-7a plays an important role in AML cell chemoresistance. This report extends the concept of targeting CXCR4 in AML by defining a complex network with the microRNA let-7a.

As an alternative to small molecular CXCR4 inhibitors, many potent peptide CXCR4 inhibitors have been identified [24] and tested in AML models. In 2011, Beider et al. [25] demonstrated that a small peptide CXCR4 inhibitor, 4F-benzoyl-TN14003 (BKT140), exhibits CXCR4-dependent preferential cytotoxicity toward leukemia and multiple myeloma in contrast to AMD3100. They found that BKT140 stimulated cell death through apoptosis in an APL cell line (NB4), and significantly reduced the growth of a subcutaneous xenograft with NB4 cell lines in SCID/beige mice. Zhang et al. [11] also reported that BKT140 alone selectively induced leukemia regression in primary AML xenograft models when AML cells expressed high CXCR4 levels and displayed a significant migratory response to CXCL12. Additionally, CXCR4 inhibition caused increased apoptosis of leukemic cells in vitro and in vivo. Both studies suggest a potential therapeutic use for BKT140 as a monotherapy. In 2015, Cho et al. [26] also demonstrated remarkable antileukemia activity of peptidic CXCR4 inhibitor, LY2510924, as a monotherapy and in combination with anti-AML chemotherapy. In vitro data revealed that LY2510924 at nanomolar concentrations rapidly and durably disrupts the CXCL12-CXCR4 axis in AML cells, which inhibits proliferation of AML cells rather than causing cell death (in contrast to BKT140 data). Using primary AML xenograft models, they found that LY2510924 causes mobilization of leukemic cells into the circulatory system, inhibits multiple prosurvival signals generated by the CXCL12/CXCR4 axis, and induces myeloid differentiation; thereby, producing antileukemia effects as monotherapy. This antileukemia activity strongly synergized with chemotherapy consisting of cytarabine and doxorubicin in xenograft models, resembling standard induction chemotherapy in human trials. In summary, preclinical data of peptidic CXCR4 inhibitors suggest promising antileukemia effects as monotherapy in addition to their chemosensitization effects. However, because the findings vary, more research is needed to explore the potential for CXCR4 inhibitors to induce cell death through apoptosis.

In recent years, several preclinical studies have reported promising antileukemia effects of anti-CXCR4 monoclonal antibodies as monotherapy. In contrast to small molecules and peptide CXCR4 inhibitors, monoclonal antibodies are expected to exert antileukemia effects through additional mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). In 2013, Kuhne et al. [27] introduced ulocuplumab (BMD-936564/MDX-1338), a fully humanized immunoglobulin G4 (IgG4) monoclonal antibody that specifically recognizes human CXCR4. They found that ulocuplumab exhibits antitumor activity in established tumors including subcutaneous xenograft models of APL and induces apoptosis on a panel of cell lines including AML. They also proposed that antibody-induced apoptosis is one of the mechanisms of tumor-growth inhibition. Another humanized anti-CXCR4 IgG4 monoclonal antibody, LY2624587, also exhibited potential for inducing apoptosis in human lymphoma and leukemia in vitro and in vivo [28]. Preclinical data for the anti-CXCR4 IgG1 monoclonal antibody, PF-06747143, were recently presented at the Annual Meeting of the American Society of Hematology; the authors suggested that CDC and ADCC are mechanisms involved in the antileukemia effect in AML cell lines [29]. PF-06747143 exerted an antileukemia effect as monotherapy in primary AML xenograft models [30]. Overall, the preclinical data, as well as the plausible additional mechanisms for AML, suggest that anti-CXCR4 monoclonal antibodies have promise in clinical applications, while also raising concerns about toxicity in the process of normal hematopoiesis.

Based on the encouraging preclinical evidence of CXCR4 inhibition in AML, a number of clinical studies involving different classes of agents have been initiated. However, the evidence for safety and efficacy is currently limited to early-phase clinical trials (Table 1). Most related clinical trials have been designed to investigate the chemosensitizing effect of AML3100 in combination with chemotherapy or targeted agents. Uy et al. [40] reported a phase 1/2 study of AMD3100 in combination with chemotherapy in relapsed/refractory AML. In the study, 52 patients were treated with AMD3100 in combination with mitoxantrone, etoposide, and cytarabine (MEC). They demonstrated approximately a 2-fold mobilization of leukemic blasts into circulation, which returned to baseline within 12 hours. There was no evidence of symptomatic hyperleukocytosis or delayed count recovery, and a complete remission (CR) and CR with incomplete count recovery (CRi) rate of 46% was achieved, which is encouraging compared to published studies of MEC alone. In a randomized phase 3 study of MEC, the overall CR rate of MEC was 21% [41]. In a frontline setting, preliminary data from a phase 1 study of AMD3100 in combination with cytarabine and daunorubicin (7 + 3 regimen) revealed that the toxicity and hematopoietic recovery expected with this 7 + 3 regimen was not significantly altered by the addition of AMD3100 [42]. The authors observed transient mobilization of AML blasts into circulation immediately following AMD3100 treatment, and achieved a CR/CRi of 76%. A study of newly diagnosed, elderly patients with AML found that a combination of AMD3100 with 10 days-decitabine regimen resulted in a 43% CR/CRi; notably, 52% of patients without previous hypomethylating agent exposure and 53% of patients with adverse cytogenetics achieved CR/CRi [43]. The authors administered AMD3100 concomitantly with decitabine in every other cycle, and the maximal tolerable dose used was 810 µg/kg without clinically significant hyperleukocytosis. Overall, these early-phase clinical data suggest that combining AMD3100 with chemotherapy or decitabine is safe and associated with favorable response rates as well as a transient mobilization of leukemic cells into circulation by AMD3100. Based on preclinical data indicating increased activity of sorafenib when combined with AMD3100 and G-CSF in FLT3-ITD mutated AML [5], Andreeff et al. [44] completed a phase 1 study testing this finding in patients with FLT3-ITD mutated relapsed/refractory AML. The authors observed a 40-fold increase in circulating blasts as a result of mobilization, with preferential mobilization of leukemic versus non-leukemic cells. A CR/CRi of 28% and a partial response of 33% were achieved, and responses included three patients refractory to previous FLT3 inhibitors. Side effects included hyperleukocytosis, hypertension, hand foot syndrome, and diarrhea, the latter two attributable to sorafenib. This striking mobilization of leukemic cells by AMD3100/G-CSF provides proof of principle among FLT3-mutated AML patients, and the preferential mobilization of AML over normal cells further supports the clinical development of this treatment option, while the on-target side effects of hyperleukocytosis should be cautiously monitored.

The peptidic CXCR4 inhibitor, BL-8040 (BKT140), has prolonged pharmacodynamic effects and exerts direct proapoptotic activity against AML cells [11,25]. In a phase 1/2 trial of patients with relapsed/refractory AML, patients received 2 days of BL-8040 as a monotherapy followed by BL-8040 and cytarabine for 5 days [45]. The preliminary results of this study suggest that all tested doses of BL-8040 are safe. Two days of treatment with BL-8040 mobilized a 40.2-fold increase in immature AML progenitors from the marrow, followed by a 58% decrease in leukemia progenitor cells in the marrow on day 3. Apoptosis induction of blasts was detected in 5/9 samples available for analysis, which was associated with a 3.1-fold increase in monocytic and granulocytic terminal differentiation. During dose escalation in 22 patients, a CR/CRi of 38% was achieved. These findings suggest that the induction of apoptosis and differentiation, as well as mobilization of AML blasts from the protective marrow, may act as mechanisms of CXCR4 inhibition. Future combination studies with BL-8040 are ongoing or planned.

Becker at al. [46] reported a phase 1 study using ulocuplumab (BMD-936564/MDX-1338), an anti-CXCR4 fully human IgG4 monoclonal antibody, which appears to have direct proapoptotic effects [27], in combination with MEC, in 73 patients with relapsed/refractory AML. Different doses of ulocuplumab were administered one week prior to starting MEC, and 3 additional weekly doses per MEC cycle thereafter, without dose-limiting toxicity. A fairly respectable CR/CRi of 51% was achieved, and, notably in four patients, CR/CRi was documented after a single dose of ulocuplumab monotherapy. Transient, mild, or moderate thrombocytopenia were the only treatment-related adverse events. A median 2- and 5-fold mobilization of leukocytes and leukemic blasts into the circulation was reported at day 8, respectively. This first clinical report of an anti-CXCR4 monoclonal antibody suggests that ulocuplumab exhibits safe and significant antileukemia activity in AML. The elimination of BM leukemia observed after a single dose of ulocuplumab in selected patients suggests a contribution from other mechanisms, such as ADCC, which may be related to the nature of the antibody. Future clinical trials with other antibody drugs, such as LY2624587 (IgG4) and PF-06747143 (IgG1), are worth considering.

Early-phase trials suggest that the addition of CXCR4 inhibitors is tolerable and appears to exhibit signs of clinical activity. Further phase 2 and 3 trials are required to determine the benefit of CXCR4 inhibitors in combination with chemotherapy or targeted agents. Despite their small number, short follow-up, and small patient populations, early-phase trials indicate that CXCR4 inhibitors are generally safe, but they may lead to an on-target adverse event of hyperleukocytosis, which should be carefully considered in future patient selection, dosing, and schedule of combinations. Another potential adverse effect is an unwanted effect on healthy HSCs, such as myelosupression, or a deleterious effect on immune cells, even though currently available clinical data have not reported a significant delay in the recovery of blood cells after chemotherapy.

Most importantly, an optimal combination and schedule is required to enhance the efficacy of CXCR4 inhibitors without increasing potential toxicity. Different pharmacokinetic and pharmacodynamic characteristics of CXCR4 inhibitors should be considered to design clinical trials, particularly for drugs with a potential to directly induce cell death, such as BL-8040 or ulocuplumab.

The main subjects for ongoing clinical studies are patients with relapsed/refractory AML of any age and newly diagnosed, elderly patients with AML, the latter presenting a huge unmet need. Another unexplored area for future research is targeting AML-niche interactions in patients in remission after conventional chemotherapy to eradicate minimally residual disease or LSCs, which are thought to be responsible for the majority of relapses [47].