Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

Multiple studies have demonstrated that interaction with the bone marrow stromal microenvironment contributes to the survival of leukemia cells. One explanation for this phenomenon is the interaction between the cell surface receptors CXCR4 and CXCL12. Through CXCL12/CXCR4-mediated chemotaxis, leukemia cells migrate to microscopic niches within the bone marrow, which leads to increased proliferation and survival. Several studies have suggested that increased CXCR4 expression may portend a poor prognosis in various types of leukemia, possibly due to increased protection of leukemia cells by bone marrow stroma. A potential therapeutic strategy to overcome this stromal-mediated survival advantage is to target CXCR4. Inhibition of CXCR4 may allow leukemia cells to be released from bone marrow niches that confer resistance to chemotherapy and negate the survival benefit imparted by bone marrow stroma.

Cure rates in pediatric acute myeloid leukemia (AML) remain suboptimal. Overexpression of the surface receptor CXCR4 is associated with poor outcome in acute lymphoblastic leukemia (ALL) and AML. Certain non-chemotherapeutic agents have been shown to modulate CXCR4 expression and alter leukemia interactions with stromal cells in the bone marrow microenvironment. Because chemotherapy is the mainstay of AML treatment, we hypothesized that standard cytotoxic chemotherapeutic agents induce dynamic changes in leukemia surface CXCR4 expression, and that chemotherapy-induced upregulation of CXCR4 represents a mechanism of acquired chemotherapy resistance. Here, we show that cell lines variably upregulate CXCR4 with chemotherapy treatment. Those that showed upregulation were differentially protected from chemotherapy-induced apoptosis when co-cultured with stroma. We further explored the functional effects of chemotherapy-induced CXCR4 upregulation in an AML cell line (MOLM-14, which consistently upregulated CXCR4) and primary samples. We found enhanced stromal-cell derived factor-1α (SDF-1α)-mediated chemotaxis and stromal protection from additional chemotherapy-induced apoptosis. Further, treatment with the CXCR4 inhibitor plerixafor preferentially decreased stromal protection in cells with higher chemotherapy-induced upregulation of surface CXCR4. Upregulation of surface CXCR4 by standard chemotherapy may represent a mechanism of chemotherapy resistance in pediatric AML and may be a biomarker that can identify optimal patients for CXCR4 inhibition.

In spite of advances in the treatment of pediatric acute lymphoblastic leukemia (ALL), a significant number of children with ALL are not cured of their disease. We and others have shown that signaling from the bone marrow microenvironment confers therapeutic resistance, and that the interaction between CXCR4 and stromal cell-derived factor-1 (SDF-1 or CXCL12) is a key mediator of this effect. We demonstrate that ALL cells that upregulate surface CXCR4 in response to chemotherapy treatment are protected from chemotherapy-induced apoptosis when co-cultured with bone marrow stroma. Treatment with the CXCR4 inhibitor plerixafor diminishes stromal protection and confers chemosensitivity. Using xenograft models of high-risk pediatric ALL, plerixafor plus chemotherapy induces significantly decreased leukemic burden, compared to chemotherapy alone. Further, treatment with plerixafor and chemotherapy influences surface expression of CXCR4, VLA-4, and CXCR7 in surviving ALL blasts. Finally, prolonged exposure of ALL blasts to plerixafor leads to a persistent increase in surface CXCR4 expression, along with modulation of surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis, findings that may have implications for therapeutic resistance. Our results suggest that while CXCR4 inhibition may prove useful in ALL, further study is needed to understand the full effects of targeting the leukemic microenvironment.

Background Plerixafor, a reversible CXCR4 antagonist, inhibits interactions between leukemic blasts and the bone marrow stromal microenvironment, and may enhance chemosensitivity. A phase 1 trial of plerixafor in combination with intensive chemotherapy in children and young adults with relapsed or refractory acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS) was performed to determine a tolerable and biologically active dose. Procedure Plerixafor was administered daily for 5 days at 4 dose levels (6, 9, 12, and 15 mg/m2/dose) followed four hours later by high-dose cytarabine (every 12 hours) and etoposide (daily). Results Nineteen patients (13 AML, 5 ALL, 1 MDS) were treated. The most common grade 3 or greater nonhematologic toxicities attributable to plerixafor were febrile neutropenia and hypokalemia. There were no dose limiting toxicities (DLTs). Plerixafor exposure increased with increasing dose levels and clearance was similar on days 1 and 5. Eighteen patients were evaluable for response. Two patients achieved complete remission (CR) and 1 patient achieved CR with incomplete hematologic recovery (CRi): all 3 had AML. No responses were seen in patients with ALL or MDS. Plerixafor mobilized leukemic blasts into the peripheral blood in 14 of 16 evaluable patients (median 3.4-fold increase), and degree of mobilization correlated with surface CXCR4 expression. Conclusions Plerixafor, in combination with high-dose cytarabine and etoposide, was well-tolerated in children and young adults with relapsed/refractory acute leukemias and MDS. While biologic responses were observed, clinical responses in this heavily-pretreated cohort were modest.

Summary Infants with MLL-rearranged (MLL-R) acute lymphoblastic leukaemia (ALL) have a dismal prognosis. While most patients achieve remission, approximately half of patients recur with a short latency to relapse. This suggests that chemotherapy-resistant leukaemia stem cells (LSCs) survive and can recapitulate the leukaemia. We hypothesized that interactions between LSCs and the bone marrow microenvironment mediate survival and chemotherapy resistance in MLL-R ALL. Using primary samples of infant MLL-R ALL, we studied the influence of bone marrow stroma on apoptosis, proliferation, and cytotoxicity induced by the FLT3 inhibitor lestaurtinib. MLL-R ALL were differentially protected by stroma from spontaneous apoptosis compared to non-MLL-R ALL. Co-culture of bulk MLL-R ALL in direct contact with stroma or with stroma-produced soluble factors promoted proliferation and cell cycle entry. Stroma also protected bulk MLL-R ALL cells and MLL-R ALL LSCs from lestaurtinib-mediated cytotoxicity. Previous studies have demonstrated that CXCR4 mediates bone marrow microenvironment signalling. Using a xenograft model of MLL-R ALL, we demonstrated that CXCR4 inhibition with AMD3100 (plerixafor) led to markedly enhanced efficacy of lestaurtinib. Therefore, the bone marrow microenvironment is a mediator of chemotherapy resistance in MLL-R ALL and targeting leukaemia-stroma interactions with CXCR4 inhibitors may prove useful in this high-risk subtype of paediatric ALL.

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Background: Inadequate myelosuppression during maintenance therapy for acute lymphoblastic leukemia (ALL) is associated with an increased risk of relapse. One mechanism is skewed metabolism of 6-mercaptopurine (6MP), a major component of maintenance therapy, which results in preferential formation of the hepatotoxic metabolite (6-methyl mercaptopurine [6MMP]) with low levels of the antileukemic metabolite, 6-thioguanine nucleotides (6TGN). Allopurinol can modify 6MP metabolism to favor 6TGN production and reduce 6MMP. Methods: Patients in maintenance were considered for allopurinol treatment who had the following features: (a) Grade ≥3 hepatotoxicity; (b) Grade ≥2 nonhepatic gastrointestinal (GI) toxicity; or (c) persistently elevated absolute neutrophil count (ANC) despite >150% protocol dosing of oral chemotherapy. Results: From 2013 to 2017, 13 ALL patients received allopurinol: nine for hepatotoxicity, five for inadequate myelosuppression, and three for nonhepatic GI toxicity (four met multiple criteria). Allopurinol was well tolerated, without significant adverse events. Allopurinol resulted in a significant decrease in the average 6MMP/6TGN ratio (mean reduction 89.1, P = .0001), with a significant increase in 6TGN (mean 550.4, P = .0008) and a significant decrease in 6MMP (mean 13 755, P = .0013). Patients with hepatotoxicity had a significant decrease in transaminase elevation after starting allopurinol (alanine transaminase [ALT] mean decrease 22.1%, P = .02), and all with nonhepatic GI toxicity had improved symptoms. Those with inadequate myelosuppression had a significant increase in the time with ANC in goal (mean increase 26.4%, P = .0004). Conclusions: Allopurinol during ALL maintenance chemotherapy is a safe, feasible, and effective intervention for those who have altered metabolism of 6MP causing toxicity or inadequate myelosuppression.