ALBERT

Description: Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with relapse rates approaching 40% in children. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is implicated in promoting many cancer phenotypes, including AML. Additionally, the important role of STAT3 in microenvironment-mediated chemoresistance is well established. Therefore, STAT3 is an important target for the development of new agents. Like other transcription factors, the structure of the STAT3 protein does not easily lend itself to the development of a small molecule inhibitor that is both potent and specific. Several commercially available and academic STAT3 inhibitors have been reported, but none is yet suitable for broad clinical application. We have developed a novel class of naphthalene sulfonamide small molecule STAT3 inhibitors with efficacy in AML cell lines and primary samples (e.g. C188-9 [Redell, et al, 2011, Blood 117:5701-9]). Rational optimization steps have yielded a new lead compound, MM-206, with improved stability in cellular contexts. Our aim is to evaluate the activity of MM-206 in preclinical AML models that include the bone marrow stromal environment. Inhibition of STAT3 activity: MM-206 potently inhibited the STAT3 SH2 domain-phosphopeptide interaction, with IC50 1.2 μM by surface plasmon resonance assays. Further, MM-206 attenuated phosphorylation of G-CSF-induced STAT3 as measured by intracellular flow cytometry. AML cell lines were treated with increasing doses of MM-206 for 30 minutes, followed by stimulation with G-CSF to induce STAT3 phosphorylation (pY705). The IC50 values for inhibition of phosphorylation were 1-3 µM, indicating potent inhibitory activity in cells. Similarly, MM-206 inhibited G-CSF-induced PIM1 transcription in a dose dependent manner in AML cell lines. Anti-leukemia activity in vitro: MM-206 demonstrated dose-dependent induction of apoptosis in AML cell lines and pediatric AML samples, even in the presence of bone marrow stromal cells. Cell lines and patient samples were cultured alone or co-cultured with mOrange-transduced HS5 or HS27A stromal cells, then treated with increasing doses of MM-206 for 24 hours. Cells were stained with Annexin V-FITC and apoptosis in the mOrange-negative cells was quantified by FACS. EC50 values were consistent with the doses for inhibition of phosphorylation, ranging from 1 - 10 µM. The increase in EC50s in the stroma co-cultured AML cells, compared to AML cells cultured alone, was ~3-fold, indicating only moderate environment-derived protection against this compound. In complementary experiments, luciferase-transduced AML cell lines were cultured alone or with stromal cells, treated with MM-206 for 24 hours, then AML cell viability was assessed by luminescence. Again, IC50s were in the range of 3 µM for all cell lines tested. There was no difference in potency for cells on stroma in the luminescence assay. In contrast to the anti-AML activity, EC50s were 〉30 µM in cell line models of acute lymphoblastic leukemia, a disease not typically associated with STAT3-dependent survival. Anti-leukemia activity in vivo: We engrafted NSG mice with luciferase-expressing MV4-11 AML cells. After 2 weeks, mice were randomized to receive MM-206 (30 mg/kg) or vehicle, ip daily Monday-Friday, for 2 or 4 weeks. MM-206 treatment delayed disease progression, as evidenced by significantly lower luminescence values (p

Description: Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704). To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children's Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p

Description: Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p

Description: Despite aggressive chemotherapy, relapse occurs in almost half of children with acute myeloid leukemia (AML), with very dismal survival. Novel and mechanism-driven therapies are desperately needed to conquer chemotherapy resistance and leukemic stemness in pediatric AML. Within the bone marrow niche, stromal cells protect leukemia cells from chemotherapy, maintain leukemic stemness, and eventually lead to disease recurrence. We developed an in vitro AML cell-stromal cell co-culture model to mimic bone marrow microenvironment. Stroma-leukemia cell interaction leads to activation of various signaling molecules in AML cells that allow them to evade apoptosis. One such example is extracellular signal-regulated kinases 1/2 (ERK1/2), important pro-survival proteins. ERK1/2 are activated by the Ras/Raf/ mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) pathway downstream of signals from the stroma. We recently showed that stromal co-culture activates ERK1/2 in pediatric AML samples, contributing to chemotherapy resistance (Long, et al, 2017, Oncotarget, 8:90037). To identify genes that are regulated in AML cells by ERK1/2 activation, 4 pediatric AML samples were cultured alone, or co-cultured with mOrange-expressing stroma for 24 hours, in the presence or absence of a selective MEK inhibitor, selumetinib (1 μM). Thereafter, cells were flow sorted to exclude mOrange+ stroma and CD45high/SSClow lymphocytes. Sorted AML cells underwent total RNA extraction for nCounter® PanCancer Pathways Panel (Nanostring Technologies) gene expression profiling study. We focused on the genes that were either up- or down-regulated by co-culture with stroma, and changed in the reverse direction by the addition of selumetinib. We chose a few genes among the list (BMP2, BNIP3, H2AFX, DUSP2, FZD3, BCL2L1, CHEK2) that are reported to be involved in oncogenesis, chemotherapy resistance, cell growth and survival. Using qRT-PCR, we confirmed bone morphogenic protein 2 (BMP2) to be upregulated in AML cells by stroma, and the effect of stroma was reversed by selumetinib. Further, we confirmed the same change of BMP2 at the protein level by FACS. Smad1, 5 and 8 are transcriptional factors immediately downstream from BMP receptors and play a central role in BMP signal transduction. Using FACS we discovered stroma-induced activation of Smad 1/8 in pediatric AML patient samples, which was partially alleviated by selumetinib and a selective BMP inhibitor, K02288 (10 μM). BMPs are growth factors that belong to the transforming growth factor beta (TGF-beta) superfamily and are thought to be involved in stem cell properties such as self-renewal. To determine if the BMP-Smad pathway plays a role in chemotherapy resistance, 4 pediatric AML patient samples were cultured on or off stromal cells, and treated with cytarabine (10 μM) with or without K02288 (10 μM) for 24h. Cells were analyzed for cytarabine-induced apoptosis with Annexin V staining by FACS, excluding stromal cells and lymphocytes. K02288 treatment did not alter cytarabine-induced apoptosis. We next tested the potential role of BMP-Smad pathway in leukemic self-renewal in pediatric AML samples. Pediatric AML patient samples were plated at 50,000/ml or 100,000/ml in Methocult medium (H-4535) to quantify stem and progenitor cells. Samples were treated with vehicle or K02288 (10 μM). Colonies and viable cells were counted and normalized to control 7-14 days after plating. Harvested cells were stained for CD34, CD14 and CD11b to evaluate differentiation by FACS. Remaining cells were replated at the same density for 2 more rounds. We found that K02288 reduced colony counts (e.g., 100±0% in vehicle control, v. 25±10% in K02288, n=5, p

Description: In B lymphoblastic leukemia (B-ALL), genome-wide association studies have revealed that deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are present in nearly 30% of patients. These lesions are most prevalent in high-risk subsets, including greater than 60% of patients with Philadelphia chromosome positive (Ph+) and Ph-like ALL. IKZF1 deletions are associated with an increased risk of relapse, therapy resistance, and inferior survival. It is therefore imperative to devise new treatment strategies for this poor-prognosis subset of patients. To this regard, using novel CRISPR-Cas9 genome editing strategies, we developed a series of human B-ALL cell lines with IKZF1 deletions. These robust model systems have allowed us to investigate the underlying biology of IKZF1-deleted B-ALL. Our studies have thus far shown that IKZF1 deletion results in a stem cell-like gene expression profile, enhanced bone marrow homing and engraftment, and cell-intrinsic chemoresistance, consistent with the relapsing disease phenotype observed in affected patients. We are using these model systems to explore possible mechanisms of chemoresistance and delineate new strategies to improve response to therapy. Global gene expression analysis of the engineered Nalm-6 IKZF1-deleted cells by RNA-seq revealed potential therapeutic vulnerabilities. IKZF1-deleted cells are characterized by increased activation of the JAK/STAT pathway with overexpression of JAK1, JAK3, STAT3, and STAT5. Aberrant activation of this pro-survival, anti-apoptosis pathway is associated with poor-prognosis leukemia; thus, we postulated this is a contributor to the chemoresistance inherent to IKZF1-deleted B-ALL. We explored the therapeutic potential of targeting the JAK/STAT pathway by treating IKZF1-deleted cells with selective inhibitors of JAK1/3 (tofacitinib) and STAT3 (MM-206) and calculated the IC50 by Annexin V/7-AAD double-negative population after 48 to 72 hours of treatment. The IKZF1 wild-type cells were sensitive to both compounds, suggesting activated JAK/STAT signaling is critical to cell survival. In comparison, the IKZF1-deleted cells were relatively resistant to both compounds (MM-206 IC50 : 5.6 µM vs. 8.2 µM, p 〈 0.001; tofacitinib IC50 : 43 nM vs. 55 nM, p = 0.05) similar to the relative resistance to ABL1-tyrosine kinase inhibition observed in Ph+ B-ALL cells with loss of function IKZF1 mutations. However, we postulated that inhibition of the JAK/STAT pathway could still augment the effects of standard chemotherapy. Indeed, whereas IKZF1-deleted Nalm-6 cells are highly resistant to glucocorticoid chemotherapy alone, when the cells were also treated with sub-IC50 levels of MM-206, we observed a significant re-sensitization to dexamethasone-induced apoptosis. A similar pattern of re-sensitization was seen with the combination of sub-IC50 MM-206 and vincristine treatment. Additionally, our gene expression analysis of the IKZF1-deleted Nalm-6 cells revealed significantly increased expression of the receptor tyrosine kinase, FLT3. Overexpression was confirmed at the protein level by flow cytometry for cell-surface FLT3. We treated our engineered cell lines with the potent and selective FLT3 inhibitor quizartinib and again found that the IKZF1-deleted cells were relatively resistant compared to the wild type cells (IC50 : 240 nM vs. 282 nM, p 〈 0.01). Postulating that parallel activation of the JAK/STAT pathway may contribute to this resistance, we treated our cells with pacritinib, a combined JAK/FLT3 inhibitor. The IKZF1-deleted cells were as sensitive to this compound as the wild type cells, suggesting dual targeting of FLT3 and JAK may be efficacious for the treatment of IKZF1-deleted B-ALL. Our data support that IKZF1-deleted B-ALL is an aggressive disease characterized by cell-intrinsic chemoresistance. We found that loss of IKAROS amplifies pro-survival, anti-apoptotic signaling pathways, a likely contributing mechanism to chemoresistance. IKZF1 deletion confers relative resistance to targeted inhibitors of these pathways. However, the combined JAK/FLT3 inhibition exhibits therapeutic efficacy. Additionally, the combination of a JAK/STAT pathway inhibitor with conventional chemotherapy including dexamethasone and vincristine may be a promising strategy to overcome the chemoresistance inherent to this poor-prognosis subset of B-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. It is clear that the bone marrow microenvironment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. We recently reported that patients whose AML blasts activated Stat3 in response to two bone marrow-derived ligands, G-CSF and IL-6, had a significantly superior survival rate, compared to patients whose blasts failed to activate Stat3 (Redell, et al, 2013, Blood121:1083). In this followup study of a subset of these patients, we further investigated the basis for the failure of the Stat3 response, as well as mechanisms of environment-mediated chemotherapy resistance. Our hypotheses were A) Stat3 responsiveness to G-CSF/IL-6 reflects the cell’s general ability to respond to ligand stimulation, rather than a specific defect in a Stat3 pathway; and B) Stat3 responsiveness correlates with other functional responses to stromal exposure, including survival, proliferation, and chemotherapy resistance. Second samples from 27 patients were studied by FACS analysis of intracellular markers. To study stroma-induced changes, AML cells were co-cultured on stromal cells overnight, or briefly stimulated with stroma-conditioned medium (CM). We used HS5 cells, which secrete high levels of many soluble factors, and HS27A cells, which secrete very few factors. The stromal cell lines express mOrange for exclusion by FACS. First, primary AML cells were stimulated with G-CSF (10 ng/ml) or IL-6 + soluble IL-6R (5 + 10 ng/ml) for 15 min, then pY-Stat3 (PE) and pERK1/2 (AlexaFluor488) were measured by FACS. Data were acquired on the LSRII (BD) and analyzed with FCSExpress4 (DeNovo). Ligand responses were expressed as the fold change in mean fluorescence intensity over unstimulated cells (ΔMFI). We found a strong correlation between G-CSF-induced pY-Stat3 ΔMFI and pERK1/2 ΔMFI (R=0.858, p

Description: Abstract 242 Overall objective: AML is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the chance of survival is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML cells, which could potentially lead to new therapies for AML. Methods, Results & Conclusions: We used two human bone marrow stromal cell lines, HS-5 and HS-27 for our studies. Both provide physical contact with AML cells, while HS-5 cells secrete many more cytokines and growth factors than HS-27 stromal cells. To verify the difference between HS-5 and HS-27 in their secreted soluble factors, both stroma-conditioned media were harvested and soluble factors were quantified by multiplex cytokine assay for 42 individual soluble factors. We detected 23 factors in HS-5 conditioned medium, including G-CSF, IL-6, and MCP-3 at very high levels. HS-27-conditioned medium contained only a few cytokines at similar levels as HS-5, e.g., VEGF and Fractalkine. Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4, THP-1 and Kasumi-1) were cultured alone or co-cultured with HS-5 or HS-27 cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours. Cells were then harvested and labeled with annexin V-FITC. Stromal cells were identifiable by stable mOrange expression, and the percentage of apoptotic AML cells (FITC positive and mOrange negative) was determined by FACS. Both HS-5 (p