Supplemental Figure 1 and Figure 2 Supplemental Tables 1-5

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.

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.

Abstract 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/m2/day (elevated lipase) and 1 of 9 had a DLT (hyperbilirubinemia) at the highest tested dose of 60 mg/m2/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/m2/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.

Background : WT1 is a zinc finger transcriptional regulator and acts as a tumor suppressor gene in various cell types. WT1 mutations are reported in approximately 10% of both adult and pediatric patients with acute myeloid leukemia (AML), and at a lower frequency in patients with myelodysplastic syndome (MDS). Reported mutations consist of insertions, deletions or point mutations, and are thought to alter WT1 DNA-binding ability and result in a loss of function. WT1 mutations are associated with FLT3/ITD mutations in AML, suggesting possible leukemogenic cooperativity, and yet WT1 mutations have been independently associated with treatment failure and a poor prognosis. Recently, a physical interaction demonstrated between WT1 and TET2 suggests a common functional pathway, and explains the mutual exclusivity of these mutations in AML. Despite these observations, the functional contribution of WT1 mutations in hematologic malignancies is not entirely understood. To our knowledge, we are the first to describe here a hematologic phenotype in a WT1 mutant mouse model and in a novel WT1 mutant x FLT3/ITD crossbred mouse model.Methods: Knock-in WT1 mutant mice are heterozygous for missense mutation R394W in the DNA-binding domain, which has been described in cases of human AML. Mice with a heterozygous 18-bp ITD knocked into the FLT3 gene were crossbred with the WT1 mutant mice, and Kaplan-Meier survival analysis was performed across genotypes. CBCs and BM cytospin morphology from moribund mutant mice were compared to wild type controls. To create a transplant model, 2e6 whole BM cells from each genotype were injected into lethally irradiated congenic mice. Competitive transplants were performed by injecting a 1:1 ratio of CD45.1 wild type (control) cells with CD45.2 WT1 mutant or wild type (test) cells into lethally irradiated C45.1 recipients.Results : We noted an expansion of lineage negative cells and various progenitor cell compartments in WT1 mutant (WT1mut) BM relative to wild type (wt); including the megakaryocyte-erythroid progenitor (MEP) compartment. WT1mut BM cells from two-month old mice showed an increased ability to serially replate in methylcellulose culture compared to wt BM cells, demonstrating aberrantly enhanced self-renewal capacity.WT1mut mice demonstrated a trend towards an inferior late survival compared to wt in survival analysis, and several moribund WT1mut mice were found to have anemia and erythrodysplasia. Most ITD mice developed a fatal myeloproliferative neoplasm (MPN), as previously described. Interestingly, double mutant mice (WT1mut+ITD) had an inferior survival compared to ITD (p <0.001) or WT1mut alone, and BM from moribund WT1mut+ITD mice demonstrated features of both erythrodysplasia and myeloproliferation, diagnostic of MDS/MPN. We then transplanted BM from each genotype into lethally irradiated congenic mice. WT1mut BM transplant recipients showed a trend toward inferior survival compared to wt BM recipients. Strikingly, all evaluable WT1mut recipient mice developed MDS in this model; manifested as decreased hemoglobin (p = 0.03), clear erythrodysplasia, and a decreased myeloid:erythroid (M:E) ratio. ITD BM recipients developed MPN as expected, and the majority of WT1mut+ITD BM recipients developed MDS/MPN. Finally, in a competitive transplant model, WT1mut BM cells exhibited an initial engraftment disadvantage, followed by a increasing engraftment advantage over wt BM cells at later time points - further supportive data that WT1mut BM causes dysfunctional hematopoiesis, a hallmark of MDS.Conclusion: BM cells from mice with the leukemogenic WT1 mutation R394W demonstrate enhanced self-renewal of hematopoietic progenitor cells, which could potentially prime cells for leukemic transformation upon acquisition of cooperative events. Importantly, transplanted mice with WT1 mutant BM consistently develop MDS, manifested as anemia and erythrodysplasia and contributing to a trend in decreased survival. Mice with both this WT1 mutation and a FLT3/ITD mutation develop a mixed MDS/MPN phenotype, which is a discrete diagnostic entity, and results in a more aggressive disease and inferior survival to mice with ITD mutations alone. These data provide new and important insights into the aberrant functional effects of WT1 mutations on hematopoiesis, and are the first to characterize the hematopoietic phenotype of a WT1 mutation in vivo .Disclosures No relevant conflicts of interest to declare.

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).

BackgroundRuxolitinib, an orally bioavailable JAK1/JAK2 inhibitor, may treat cancers with CRLF2 and/or JAK pathway mutations.ProcedureA 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 50mg/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).ResultsForty-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.ConclusionRuxolitinib was well tolerated in children with refractory cancer. The recommended phase 2 dose for continuous BID oral administration is 50mg/m(2)/dose. Subsequent evaluation of ruxolitinib in combination with cytotoxic chemotherapy in children, adolescents, and young adults with JAK-mutant leukemias is planned. Pediatr Blood Cancer 2015;62:1717-1724. (c) 2015 Wiley Periodicals, Inc.

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.

Abstract Introduction The MLL gene is rearranged (MLL-r) in 80% of infants with B-lymphoblastic leukemia (B-ALL). MLL-r infant B-ALL has a poor prognosis, with 4-year event-free survival less than 45%. The typical pattern of failure in MLL-r infant ALL is successful remission induction followed by early relapse, suggesting rapid emergence and/or selection of one or more chemoresistant subclones. The markedly lower observed remission induction rates at relapse (<40%) vs. diagnosis (>90%) are consistent with this hypothesis. Genomic studies of MLL-r B-ALL have revealed a striking paucity of cooperating genomic abnormalities compared to other subsets of B-ALL, which suggests heritable epigenetic changes may drive leukemogenesis, chemoresistance and evolution of relapse in MLL-r B-ALL. MLL, its fusion partners and various components of its large complexes have functional domains with known or suspected epigenetic activity. Thus, MLL rearrangement in B-ALL may trigger chromatin modifications and DNA CpG methylation changes that interplay to disrupt normal gene transcription and expression. We hypothesized that infant MLL-r B-ALL cells dynamically acquire heritable DNA CpG methylation changes, some of which may contribute to chemoresistance and evolution of relapse. To test this hypothesis we performed whole-genome bisulfite sequencing (WGBS) using paired diagnosis-relapse (DX-RL) samples to identify methylation changes that may drive relapse. Methods We evaluated paired DX-RL specimens with >95% leukemic blasts from two infants with B-ALL harboring MLL-ENL fusions: A 4 month-old female who relapsed after 1 year (case a), and an 8 month-old male who relapsed after 5 months (case b). We prepared WGBS libraries and ran paired-end sequencing (2x100bp) using Illumina HiSeq at average 25X coverage. We used Bismark for aligning reads and calling CpG methylation states. We developed an analysis pipeline to convert CpG states into CpG haplotypes, detect methylation changes at the haplotype level between DX and RL, annotate methylation changes to genes and promoters, and perform gene set enrichment analysis (GSEA) on these genes. Results First, we filtered reads falling into repeated DNA. We then obtained methylation calls at 15.6/14.3 (DX/RL) million CpGs in case (a) and 15.5/15.6 (DX/RL) million CpGs in case (b). We extracted uniquely aligned reads covering 4 contiguous CpGs, which defined a “4 CpG-site”, or “site”. We required that each site in both DX and RL is covered by at least 4 reads, which yielded 26,747 and 85,174 sites for (a) and (b). We compared the methylation level at each site in DX versus RL (e.g. a “1-0-1-1” 4 CpG-site has a methylation level of 0.75). For (a), 165 sites showed a 50% increase in methylation level at relapse (e.g., from 0.2 to 0.7), and 468 showed a 50% decrease in methylation level at relapse. For (b) there were 605 increased and 57 decreased. The 633 methylation changes in (a) mapped to 175 genes (2kb upstream to transcription end site) and the 662 changes in (b) mapped to 135 genes. Some of these genes are known to be involved in MLL-r ALL [e.g., IKZF1, in (a) and TLX2 in (b)]. GSEA was performed independently using the 175 and 135 genes in (a) and (b), respectively. Interestingly, there was substantial overlap: The top 3 gene sets for (b) (FDR q-value ≤ 2.9E-8) were also within the top 9 sets for (a) (FDR ≤ 1.1E-5). Strikingly, all 3 of these sets involved components of the polycomb repressive complex 2 (PRC2) (1. EED target genes, 2. H3K27me3 bound genes and 3. Genes increased after EZH2 knockdown). Also in common was the KEGG gene set for antigen processing and presentation [FDR 3.4E-6 in (a) and 1.7E-4 in (b)]. Conclusion WGBS allows an in-depth look at the epigenetic state of leukemic cell populations. While >98% of the methylome remained relatively stable between diagnosis and relapse (< 50% change), the hundreds of differentially methylated sites may be sufficient to influence expression of key genes and drive selection during the evolution of relapse. In addition, the ~15 million CpG methylation states are stably inherited yet variable, and comprise a rich source of information that can be used to identify evolving subclones, particularily in MLL-r ALL given its silent genomic landscape. Validation of the functional significance of the methylation changes using RNA-seq and identification of functionally important epigenetically-defined subclones is underway. Disclosures No relevant conflicts of interest to declare.

Background: Interaction between surface receptor CXCR4 (s-CXCR4) and chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow (BM) microenvironment. We previously demonstrated: 1) chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) and ALL enhances stromal protection from chemotherapy-induced apoptosis; 2) the FDA-approved CXCR4 inhibitor plerixafor reverses stromal protection and chemotherapy resistance both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged (MLL-R) ALL; 3) the novel Protein Epitope Mimetic POL5551, a selective and potent antagonist of CXCR4, blocks the SDF-1-binding site of CXCR4, inhibits SDF-1-induced chemotaxis, and reverses stromal-mediated protection from chemotherapy in vitro in pre-B and T ALL cell lines. Here, we further characterize the effects of POL5551 (POL) on surface adhesion molecule expression in ALL, and its in vivo effects in a xenograft model of HR pediatric ALL.

Background: The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. WT1 mutations are reported in approximately 10-15% of both adult and pediatric patients with acute myeloid leukemia (AML), and have been associated with treatment failure and a poor prognosis. Reported mutations consist of insertions, deletions or point mutations; and occur primarily in exon 7 or exon 9 of the WT1 gene. These mutations are thought to alter WT1 DNA-binding ability or result in a loss of function. Despite these observations, the functional contribution of WT1 mutations in leukemogenesis is still largely undetermined.

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.

Abstract Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract Background FLT3 is expressed in most human acute leukemias. When activated by FL, wild type (wt) FLT3 dimerizes and initiates downstream signals that result in proliferation and inhibition of apoptosis and differentiation. Activating FLT3 mutations (internal tandem duplications (ITDs) or point mutations) are common in AML and rare in ALL. ITD mutations confer a poor outcome in AML. In vitro, mutant FLT3 signaling can be further enhanced by binding of FL. Peripheral blood (PB) plasma FL levels rise in adults with AML, peaking about two weeks after initiation of chemotherapy. We sought to determine plasma levels of FL in pediatric patients after chemotherapy, and the functional effect of various levels of FL on both wt and mutant FLT3 leukemia cells. Methods FL levels were measured using FL ELISA on plasma samples (n=352) isolated from PB of children (n=75) enrolled on 4 multi-center acute leukemia clinical trials. Functional studies were performed on AML and ALL cell lines with wt FLT3 (HL60, RS4;11, SEMK2, and KOPN-8) and mutant FLT3 (MOLM14, MV4-11, and HB-1119). 72 hr etoposide IC50 was determined by WST-1 for each line. Cells were plated (250,000 cell/mL) for 72hr at etoposide IC50 in RPMI 1640 along with increasing concentrations of recombinant human FL (62.5 to 4,000 pg/ml). Cell cycle and apoptosis were analyzed using propidium iodide staining and annexin V/7-AAD binding, respectively. To explore the mechanism of FL effects, Ba/F3-ITD cells were incubated for 72hr in serum-free conditions with either 4,000 pg/mL (“high”), 62.5 pg/mL (“low”), or no FL. After washing, total and phosphorylated FLT3 protein levels were determined by Western blot. Results Pediatric patients receiving chemotherapy for the treatment of acute leukemia demonstrate a pattern of plasma FL rise with low levels at baseline (mean 41 pg/ml) and peak levels at day 11-14 following initiation of therapy (mean: 1,190 pg/mL; max: 5,783 pg/mL)(Fig 1A). Cell lines with FLT3 activating mutations selectively demonstrate resistance to etoposide-induced apoptosis (Fig 1B) and G2/M cell cycle arrest (Fig 1C) at low concentrations of FL (62.5 pg/mL). Dose-dependent reduction of etoposide resistance is seen with increasing concentrations of FL up to 4,000 pg/mL, suggesting that optimal etoposide-induced killing of FLT3-mutant leukemias may occur when FL plasma levels are at their peak. Ba/F3-ITD cells pre-incubated with peak concentrations of FL showed diminished baseline FLT3 phosphorylation, suggesting that the interaction of FL and FLT3/ITD exhibits substrate inhibition kinetics and results in a loss of FLT3/ITD-induced activation with high level FL exposure, thus providing a mechanistic basis for the observed loss of etoposide resistance. Conclusions Plasma FL rises to peak levels 11-14 days after initiation of chemotherapy. Through substrate inhibition of mutant FLT3 enzymatic activity, peak FL levels may reduce the etoposide resistance that characterizes FLT3-mutant leukemia cells exposed to pre-chemotherapy levels of FL. Thus, introduction of etoposide in a “time sequential” manner during periods of peak plasma FL levels may enhance killing of residual chemoresistant FLT3-mutant leukemia cells. Disclosures: No relevant conflicts of interest to declare.

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. Mol Cancer Res; 11(9); 1004–16. ©2013 AACR.

Abstract Background The protection afforded to leukemic blasts by the bone marrow microenvironment has been identified as an important mechanism of chemoresistance. Interaction between stroma-derived growth factor-1 alpha (SDF-1α) and its receptor, CXC chemoreceptor 4 (CXCR4) are implicated in chemotaxis, homing, and survival/apoptosis of normal and malignant hematopoietic cells in the bone marrow. Preclinical data demonstrates that plerixafor (AMD3100), a CXCR4 antagonist, disrupts tumor-stroma interactions and mobilizes leukemia cells from their protective stromal environment. Combinations of CXCR4 antagonists with chemotherapy have demonstrated preclinical synergy. Chemosensitization using plerixafor prior to cytotoxic chemotherapy has been tested in adults with acute leukemia. We report the first Phase I study of plerixafor (NCT01319864) delivered prior to chemotherapy in children with relapsed/refractory acute leukemia and MDS. Study Design Patients > 3 and < 30 years of age with relapsed or refractory AML, ALL, MDS or mixed phenotype acute leukemia were eligible for enrollment. Plerixafor was administered intravenously (IV) once daily followed 4 hours later by cytarabine (1 gm/m2 every 12 hours) and IV etoposide (150 mg/m2 daily) for a total of 5 days of therapy. Plerixafor pharmacokinetic studies were performed on days 1 and 5. Correlative biology studies included measurement of peripheral blood mobilization of leukemic blasts by flow cytometry, quantitative expression of CXCR4 on leukemic blasts, and the change in surface expression of CXCR4 on residual blasts after course 1 of therapy. Results Eighteen evaluable patients (11 AML, 6 ALL, 1 MDS) were treated at 4 dose levels of plerixafor (6, 9, 12, and 15 mg/m2/dose) utilizing a Rolling 6 design. The median number of prior regimens was 2.8 (range 1-7) for ALL and 2.1 (range 1-4) for AML. Six patients had high risk cytogenetics (3 ALL, 2 AML, 1 MDS). Three patients with ALL and 4 with AML had prior hematopoietic stem cell transplant (HSCT). Toxicities were consistent with intensive relapsed leukemia regimens. The most common Grade 1 and 2 toxicities attributed to plerixafor occurring in >10% of patients were anorexia, nausea, vomiting, diarrhea, fatigue, and dizziness. There were no dose limiting toxicities and no delay in count recovery attributable to plerixafor. There were responses in 3 (2 complete response (CR), 1 complete response with incomplete hematologic recovery (CRi)) of 11 AML patients (27%) and no responses in those with ALL or MDS. Peripheral leukemia-specific blast counts (measured by flow cytometry before and 4 hours after the first dose of plerixafor) demonstrated mobilization of leukemic blasts in 14 of 16 patients with samples available, with median fold increase of 3.4 (range 1.3 to 17). The degree of leukemic blast mobilization correlated positively with quantitative leukemia blast surface CXCR4 protein expression (expressed as median fluorescence index relative to isotype control), with a Pearson’s correlation co-efficient of 0.56, p=0.02. Mean ± SD plerixafor AUC values at 12 and 15 mg/m2 were 5074 ± 380 and 5732 ± 573 ng*h/mL, respectively. Drug clearance was similar between days 1 and 5 (p=0.195). Conclusion The favorable safety profile of plerixafor and biologic rationale demonstrated in this clinical trial support further clinical study of chemosensitization using CXCR4 antagonists in overcoming chemoresistance. Disclosures: Off Label Use: Plerixafor is not approved for chemosensitization in the treatment of acute leukemia.

Abstract Abstract 3605 Background: AC220 is a novel class III receptor tyrosine kinase (RTK) inhibitor that is potent and highly selective for mutant and wild type (WT) FLT3 and other class III RTK's including KIT, CSF1R, RET and PDGFR. In childhood acute myeloid leukemia (AML), ∼18% of children have FLT3 internal tandem duplication mutations (FLT3-ITD), and ∼10% high WT FLT3 expression. FLT3-ITD is associated with poor prognosis. In childhood acute lymphoblastic leukemia (ALL), the highest levels of FLT3 mRNA expression occur in cases of infants (80%) and childhood ALL with MLL rearrangements (MLL-r) (5%), both conferring poor prognosis.1,2 Study Design: TACL 2009–004is a first-in-children study using AC220 in combination with cytarabine and etoposide. Children > 1 month and < 21 years of age with relapsed/refractory AML or MLL-rearranged ALL are eligible. A standard 3+3 dose escalation design is utilized. The three doses tested (25, 40 and 60 mg/m2/day) are significantly lower than those tested in adults. Dose escalation past 60 mg/m2 occurs only if adequate biologic activity as determined by a plasma inhibitory assay (PIA) is not achieved. Intravenous (IV) cytarabine (1 gm/m2/dose every 12 hours) and IV etoposide (150 mg/m2/dose daily) are given over 5 days. AC220 is administered once daily as an oral solution on days 7–28. Patients can receive up to 2 courses of therapy. PIA testing is performed at trough time points weekly during exposure to AC220 to determine biologic activity. Results: To date, 13 patients (pts) were enrolled and 12 are evaluable for toxicity and response. One pt died from infectious complications (not drug-related) after a single dose of AC220 and was replaced. Median age at study entry was 10.2 years (range 11 mo – 20 yrs), average number of prior regimens was 2.8 (range 1–5), and 5 pts had prior stem cell transplant. Nine pts had relapsed AML, 2 had relapsed MLL-r ALL, and 1 had secondary AML. Of patients with AML, 4 had FLT3-ITD mutations and one had a D835 mutation. Toxicities were consistent with intensive relapsed leukemia regimens. Across all dose levels, non-hematologic toxicities ≥ grade 3 attributed to AC220 included vomiting (n=1), elevated transaminases (n=1), anorexia (n=2), and infection (n=3). One pt experienced a dose-limiting toxicity (DLT) on dose level 2 (40 mg/m2/day) of recurrent grade 3 elevated lipase. Dose level 2 was expanded to 6 pts without additional DLTs. Of 3 pts treated at 60 mg/m2/day, there have been no DLTs. Near total (>99%) inhibition of FLT3 phosphorylation by PIA is seen in every patient across all dose levels. Of 12 pts evaluable for response to date, 1 patient achieved a complete response (CR), 3 achieved complete response with incomplete neutrophil and platelet recovery (CRi), 5 had stable disease (SD), and 3 had progressive disease (PD). Responses in the 4 FLT3-ITD pts include 1 CR, 2 CRi and 1 SD. The FLT3-ITD patient with SD had reduction in marrow blasts without peripheral blood count recovery. An additional 6 pts will be enrolled at 60 mg/m2/day to complete safety evaluation and confirm biologic activity. Conclusions: AC220 plus intensive chemotherapy is well tolerated at doses up to 60 mg/m2/day with near complete inhibition of FLT3 phosphorylation in all pts tested to date. Response rates to date in pre-treated children with relapsed FLT3-ITD AML are encouraging. Disclosures: Off Label Use: AC220 in relapsed/refractory pediatric acute leukemia. Gammon:Ambit Biosciences: Employment.

Abstract Background We have previously demonstrated that inhibition of CXCR4 in ALL decreases CXCR4 antibody binding, inhibits SDF-1α-(CXCL12)-induced chemotaxis, and overcomes chemotherapy resistance conferred by the bone marrow microenvironment. Specifically, we found that treatment with plerixafor and araC significantly decreased leukemic burden in a xenograft model of infant ALL, compared to treatment with araC alone. In those experiments, we treated mice on 3 consecutive days per week for 2 weeks with plerixafor and araC. However, the combination did not eradicate the leukemia in our model. We hypothesized that extended exposure to plerixafor may have led to increased interactions between surviving leukemic blasts and the bone marrow microenvironment. In our current experiments, we sought to characterize the effects of prolonged exposure to plerixafor in ALL. Methods/Results We treated pre-B (HB-1119, Nalm-6) and T (CCRF-CEM-1301, Jurkat) ALL cell lines with a dose range of plerixafor and harvested cells for FACS over an extended time course. We measured surface CXCR4 (s-CXCR4) expression using 3 antibodies: 12G5, which attaches to the SDF-1α and drug-binding site of CXCR4, and 1D9 and 2B11, which do not compete with SDF-1α or drug binding. 12G5 binding was decreased by plerixafor even at 1 hour and this effect was concentration-dependent. Interestingly, we found a time and dose-dependent increase in 1D9 and 2B11 antibody binding, suggesting that plerixafor caused an actual increase in s-CXCR4 over time. Increases in 1D9 and 2B11 binding were inversely proportional to decreases in 12G5 binding. We also measured surface expression of CD49d (VLA-4), which binds to fibronectin and VCAM-1; CXCR7, which binds to SDF-1α and CXCL11; and CXCR3, which binds to CXCL9, 10, and 11. We hypothesized that CXCR4 inhibition would lead to upregulation of parallel pathways of leukemia-stroma interactions. CD49d was highly expressed at baseline, while CXCR7 and CXCR3 were expressed to a lesser degree. Treatment with plerixafor led to dose-dependent increases in CXCR7 and variable changes in CD49d and CXCR3 surface expression, suggesting that plerixafor can modulate surface expression of adhesion molecules other than CXCR4. Next, we treated ALL cell lines with plerixafor (0, 10, 100 nM) for 72 hours, washed with PBS, and resuspended the cells in fresh medium to determine the effects of extended exposure to plerixafor and subsequent withdrawal. First, we measured surface expression of s-CXCR4 after 72 hours of treatment with plerixafor and found that 12G5 binding was decreased, while 1D9/2B11 binding was increased in an inversely proportional manner. After withdrawal, 12G5 binding increased to untreated levels between 4 and 24 hours, while 1D9/2B11 binding decreased to untreated levels between 4 and 72 hours. We also measured surface expression of CD49d, CXCR7, and CXCR3 and found that the effects of plerixafor treatment and withdrawal were variable by cell line. For example, after plerixafor treatment, surface expression of CD49d and CXCR7 was increased in Nalm-6 and surface expression of CXCR7 and CXCR3 was increased in CCRF-CEM-1301. Interestingly, 4 hours after plerixafor withdrawal, CD49d expression was increased in Jurkat and Nalm-6, and CXCR7 expression was increased in CCRF-CEM-1301, HB-1119, and Jurkat. Finally, we measured migration of washed cells from each treatment condition through a permeable membrane toward medium containing SDF-1α or medium alone. Despite CXCR4 inhibition for 72 hours, all plerixafor-treated cells migrated in response to SDF-1α. In addition, some plerixafor-treated cells exhibited significantly increased SDF-1α-induced chemotaxis compared to control-treated cells. These findings imply that increases in s-CXCR4 induced by 72 hours of treatment with plerixafor are functional. Conclusions Treatment of ALL cell lines with plerixafor led to a dose-dependent decrease in 12G5 antibody binding with a simultaneous overall increase in s-CXCR4 expression. Prolonged exposure to plerixafor led to increased s-CXCR4 expression that persisted for up to 72 hours after drug withdrawal, modulated surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis. Therefore, additional careful studies of CXCR4 inhibitors and other microenvironment-targeted agents must be performed in order to determine their optimal use in ALL. Disclosures: No relevant conflicts of interest to declare.