Knock-in of the Wt1 R394W mutation causes MDS and cooperates with Flt3/ITD to drive aggressive myeloid neoplasms in mice.

Wilms tumor 1 (WT1) is a zinc finger transcriptional regulator, and has been implicated as both a tumor suppressor and oncogene in various malignancies. Mutations in the DNA-binding domain of the WT1 gene are described in 10-15% of normal-karyotype AML (NK-AML) in pediatric and adult patients. Similar WT1 mutations have been reported in adult patients with myelodysplastic syndrome (MDS). WT1 mutations have been independently associated with treatment failure and poor prognosis in NK-AML. Internal tandem duplication (ITD) mutations of FMS-like tyrosine kinase 3 (FLT3) commonly co-occur with WT1-mutant AML, suggesting a cooperative role in leukemogenesis. The functional role of WT1 mutations in hematologic malignancies appears to be complex and is not yet fully elucidated. Here, we describe the hematologic phenotype of a knock-in mouse model of a Wt1 mutation (R394W), described in cases of human leukemia. We show that Wt1 +/R394W mice develop MDS which becomes 100% penetrant in a transplant model, exhibit an aberrant expansion of myeloid progenitor cells, and demonstrate enhanced self-renewal of hematopoietic progenitor cells in vitro. We crossbred Wt1 +/R394W mice with knock-in Flt3 +/ITD mice, and show that mice with both mutations (Flt3 +/ITD/Wt1 +/R394W) develop a transplantable MDS/MPN, with more aggressive features compared to either single mutant mouse model.

A phase 1 study of the CXCR4 antagonist plerixafor in combination with high-dose cytarabine and etoposide in children with relapsed or refractory acute leukemias or myelodysplastic syndrome: A Pediatric Oncology Experimental Therapeutics Investigators' Consortium study (POE 10-03).

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 four dose levels (6, 9, 12, and 15 mg/m2 /dose) followed 4 hr later by high-dose cytarabine (every 12 hr) and etoposide (daily). RESULTS: Nineteen patients (13 with AML, 5 with ALL, 1 with 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 one patient achieved CR with incomplete hematologic recovery (CRi): all three 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 the 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.

A Phase I Study of Quizartinib Combined with Chemotherapy in Relapsed Childhood Leukemia: A Therapeutic Advances in Childhood Leukemia & Lymphoma (TACL) Study.

PURPOSE: To determine a safe and biologically active dose of quizartinib (AC220), a potent and selective class III receptor tyrosine kinase (RTK) FLT3 inhibitor, in combination with salvage chemotherapy in children with relapsed acute leukemia. EXPERIMENTAL DESIGN: Quizartinib was administered orally to children with relapsed AML or MLL-rearranged ALL following 5 days of high-dose cytarabine and etoposide (AE). A 3+3 dose escalation design was used to identify a safe and biologically active dose. Plasma inhibitory assay (PIA) testing was performed weekly to determine biologic activity. RESULTS: Toxicities were consistent with intensive relapsed leukemia regimens. One of 6 patients experienced a dose-limiting toxicity (DLT) at 40 mg/m(2)/day (elevated lipase) and 1 of 9 had a DLT (hyperbilirubinemia) at the highest tested dose of 60 mg/m(2)/day. Of 17 response evaluable patients, 2 had complete response (CR), 1 complete response without platelet recovery (CRp), 1 complete response with incomplete neutrophil and platelet recovery (CRi), 10 stable disease (SD), and 3 progressive disease (PD). Of 7 FLT3-ITD patients, 1 achieved CR, 1 CRp, 1 Cri, and 4 SD. FLT3-ITD patients, but not FLT3 wild-type (WT) patients, had significantly lower blast counts post-quizartinib. FLT3 phosphorylation was completely inhibited in all patients. CONCLUSIONS: Quizartinib plus intensive chemotherapy is well tolerated at 60 mg/m(2)/day with near complete inhibition of FLT3 phosphorylation in all patients. The favorable toxicity profile, pharmacodynamic activity, and encouraging response rates warrant further testing of quizartinib in children with FLT3-ITD AML. Clin Cancer Res; 22(16); 4014-22. ©2016 AACR.

POL5551, a novel and potent CXCR4 antagonist, enhances sensitivity to chemotherapy in pediatric ALL.

The importance of the cell surface receptor CXCR4 and the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is well-established in normal and malignant hematopoiesis. The Protein Epitope Mimetic POL5551 is a novel and potent antagonist of CXCR4. POL5551 efficiently mobilizes hematopoietic stem and progenitor cells, but its effects in acute lymphoblastic leukemia (ALL) have not been reported. Here, we demonstrate that POL5551 is a potent antagonist of CXCR4 in pre-B and T cell ALL cell lines and pediatric ALL primary samples. POL5551 has activity at nanomolar concentrations in decreasing CXCR4 antibody binding, blocking SDF-1α-mediated phosphorylation of ERK1/2, inhibiting SDF-1α-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy. POL5551 is significantly more effective at inhibiting CXCR4 antibody binding than the FDA-approved CXCR4 inhibitor plerixafor in ALL cell lines and primary samples. We also show that treatment with POL5551 in vitro and cytarabine +/- POL5551 in vivo modulates surface expression of adhesion molecules, findings that may guide the optimal clinical use of POL5551. Finally, we demonstrate that POL5551 increases sensitivity to cytarabine in a xenograft model of a high-risk pediatric ALL, infant MLL-rearranged (MLL-R) ALL. Therefore, disruption of the CXCR4/SDF-1 axis with POL5551 may improve outcomes in children with high-risk ALL.

A phase 1 dosing study of ruxolitinib in children with relapsed or refractory solid tumors, leukemias, or myeloproliferative neoplasms: A Children's Oncology Group phase 1 consortium study (ADVL1011).

BACKGROUND: Ruxolitinib, an orally bioavailable JAK1/JAK2 inhibitor, may treat cancers with CRLF2 and/or JAK pathway mutations. PROCEDURE: A phase 1 trial of ruxolitinib was performed to determine the maximum tolerated or recommended phase 2 dose, dose-limiting toxicities (DLTs), pharmacokinetics (PK), and pharmacodynamics (PD) in children with recurrent/refractory solid tumors (STs). Ruxolitinib was administered twice daily (BID) in 28-day cycles at five dose levels (15, 21, 29, 39, and 50 mg/m(2)/dose). PK and PD studies were performed during cycle 1. Toxicity, preliminary efficacy, and PK/PD were also assessed in children with relapsed/refractory hematologic malignancies (HMs). RESULTS: Forty-nine patients were enrolled, 28 with STs (dose escalation cohort) and 21 with HMs. Ruxolitinib was well-tolerated with one DLT per cohort of six patients at dose levels (DLs) 2-5. One patient with an ST had grade 5 multi-organ failure at DL2. One patient each at DL3 and DL4 had a grade 4 neutropenia, and one patient at DL5 had a grade 4 creatinine phosphokinase elevation. No objective responses were observed in patients with STs. One patient with polycythemia vera achieved a partial response and received 18 cycles of ruxolitinib. The PK of ruxolitinib were similar to that in adults. Partial inhibition of phosphorylated JAK2, STAT5, and S6 was observed in in vitro plasma inhibitory activity PD assay. CONCLUSION: Ruxolitinib was well tolerated in children with refractory cancer. The recommended phase 2 dose for continuous BID oral administration is 50 mg/m(2)/dose. Subsequent evaluation of ruxolitinib in combination with cytotoxic chemotherapy in children, adolescents, and young adults with JAK-mutant leukemias is planned.

Plerixafor as a chemosensitizing agent in pediatric acute lymphoblastic leukemia: efficacy and potential mechanisms of resistance to 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.

NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease.

Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.

Dynamic chemotherapy-induced upregulation of CXCR4 expression: a mechanism of therapeutic resistance in pediatric AML.

UNLABELLED: Cure rates in pediatric acute leukemias remain suboptimal. Overexpression of the cell-surface chemokine receptor CXCR4 is associated with poor outcome in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Certain nonchemotherapeutic 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, it was 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 therapeutic resistance. Here, it was shown that cell lines variably upregulate CXCR4 with chemotherapy treatment. Those that showed upregulation were differentially protected from chemotherapy-induced apoptosis when cocultured with stroma. The functional effects of chemotherapy-induced CXCR4 upregulation in an AML cell line (MOLM-14, which harbors consistent upregulated CXCR4) and clinical specimens were explored. Importantly, enhanced stromal-cell derived factor-1α (SDF1A/CXCL12)-mediated chemotaxis and stromal protection from additional chemotherapy-induced apoptosis was found. Furthermore, treatment with plerixafor, a CXCR4 inhibitor, preferentially decreased stromal protection with higher chemotherapy-induced upregulation of surface CXCR4. Thus, increased chemokine receptor CXCR4 expression after treatment with conventional chemotherapy may represent a mechanism of therapeutic resistance in pediatric AML. IMPLICATIONS: CXCR4 may be a biomarker for the stratification and optimal treatment of patients using CXCR4 inhibitors.

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

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 programs. 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 states. 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.