Acute myeloid leukemia (AML) is a molecularly heterogenous hematological malignancy, with one of the most common mutations being internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (FLT3). Despite the development of FLT3-directed tyrosine kinase inhibitors (TKI), relapse and resistance are problematic, requiring improved strategies. In both patient samples and cell lines, FLT3-ITD raises levels of reactive oxygen species (ROS) and elicits an antioxidant response which is linked to chemoresistance broadly in AML. NF-E2–related factor 2 (NRF2) is a transcription factor regulating the antioxidant response including heme oxygenase -1 (HO-1), a heat shock protein implicated in AML resistance. Here, we demonstrate that HO-1 is elevated in FLT3-ITD-bearing cells compared to FLT3-wild type (WT). Transient knockdown or inhibitor-based suppression of HO-1 enhances vulnerability to the TKI, quizartinib, in both TKI-resistant and sensitive primary AML and cell line models. NRF2 suppression (genetically or pharmacologically using brusatol) results in decreased HO-1, suggesting that TKI-resistance is dependent on an active NRF2-driven pathway. In AML-patient derived xenograft (PDX) models, brusatol, in combination with daunorubicin, reduces leukemia burden and prolongs survival. Cumulatively, these data encourage further development of brusatol and NRF2 inhibition as components of combination therapy for refractory AML.

Amongst the epigenetically targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in ALL. The HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels of intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations were evaluated. Both combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction. A redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.

Approximately 30% of patients with acute myeloid leukemia (AML) harbor activating mutations in the fms-like tyrosine kinase receptor 3 (FLT3). Such mutations are associated with increased propensity to relapse and dramatically decreased survival. FLT3 tyrosine kinase inhibitors (TKI), such as quizartinib, have shown modest clinical effects as single agents, and resistance remains a significant clinical problem. In addition to constitutive activity of the FLT3 receptor, AML cells with an internal tandem duplication of FLT3 (FLT3-ITD) exhibit elevated levels of reactive oxygen species (ROS). Antioxidants such as heme-oxygenase 1 (HO-1) are often upregulated in conjunction with increased ROS levels. In addition to its role as an antioxidant, HO-1 also has known proliferative and anti-apoptotic functions in some cell types. A study in non-small cell lung carcinoma recently suggested that HO-1 inhibits the Notch pathway through binding the Notch receptor. As Notch signaling exerts pro-apoptotic effects in AML, targeting HO-1 may induce Notch signaling, representing a potential therapeutic approach.

Tyrosine kinase inhibitors (TKI) have improved CML response rates, and some are effective against resistance-promoting point mutations in BCR-ABL1. However, in the absence of point mutations, resistance still occurs. Here, we identify a novel pathway mediating resistance which connects p47phox, the organizer subunit of NADPH oxidase-2 (NOX2), with early growth response-1 (Egr-1) and the Src family kinase Fyn. We found up-regulation of p47phox, Egr-1, and Fyn mRNA and protein using paired isogenic CML cell lines and mined data. Isolation of CD34+ cells and tissue microarray staining from blast crisis CML patients confirmed in vivo over-expression of components of this pathway. Knockdown studies revealed that p47phox modulated reactive oxygen species and Egr-1 expression, which, in turn, controlled Fyn expression. Interestingly, Fyn knockdown sensitized TKI-resistant cells to dasatinib, a dual BCR-ABL1/Src inhibitor. Egr-1 knockdown had similar effects, indicating the utility of targeting Fyn expression over activation. Pointedly, p47phox knockdown also restored TKI-sensitivity, indicating that targeting the NOX2 complex can overcome resistance. The NOX2/Egr-1/Fyn pathway was also conserved within TKI-resistant EGFRΔIII-expressing glioblastoma and patient-derived glioblastoma stem cells. Thus, our findings suggest that targeting the NOX2/Egr-1/Fyn pathway may have clinical implications within multiple cancer types; particularly where efficacy of TKI is compromised.

Abstract Acute myelogenous leukemia (AML) afflicts ∼12,330 new patients per year in the United States. Regrettably, only 25% of patients will survive five years past diagnosis. The most common mutation in AML is internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (Flt3), which renders it constitutively active. This mutation correlates with poor clinical prognosis and has been targeted therapeutically. Unfortunately, Flt3-directed tyrosine kinase inhibitors (TKI) have shown only modest benefit as single agents. Additionally, relapse and resistance are major factors in the treatment of Flt3-ITD+AML. In addition to its prominent role in regulating proliferative signaling, Flt3-ITD also increases production of reactive oxygen species (ROS) which act as secondary messengers, mediating oncogenesis and drug resistance. Heme oxygenase 1 (HO-1) is a ROS-responsive antioxidant that mediates proliferation and drug resistance in some cancer types. Initial analysis of patient samples from the AML TCGA cohort suggests that HO-1 is up-regulated in a subset of AML patients where its expression correlates with poor prognosis. Interestingly, HO-1 over-expression co-occurs with Flt3 receptor alterations. Thus, we hypothesized that Flt3-ITD-dependent signaling and ROS production increase HO-1, resulting in proliferation and drug resistance in AML. Constitutive expression of HO-1 protein and mRNA was elevated in murine (BaF3/Flt3-ITD) and human (MOLM13 and MV4.11) ITD+ cell lines as compared to Flt3-WT cells. This expression was dependent on Flt3-kinase, as treatment with Flt3-directed TKIs (quizartinib or lestaurtinib) attenuated HO-1 expression. Further, oxygen consumption and respiration analyses, as well as chemical inhibitors revealed a role for non-mitochondrial ROS producing enzymes in HO-1 expression in ITD+ cells. To determine a functional role for HO-1, we inhibited HO-1 with zinc protoporphyrin (ZnPP) or utilized siRNA knockdown of HO-1, both of which resulted in decreased proliferation of ITD+ cells. To determine the contribution of HO-1 in resistance to Flt3-directed TKI, we created a model of acquired resistance to Flt3-directed TKI (ITDR). HO-1 expression was elevated in ITDR compared to parental cells. However, HO-1 was no longer under control of Flt3-kinase signaling, as treatment with Flt3-directed TKI had no effect on HO-1. Similar to parental cells, knockdown of HO-1 or treatment with ZnPP resulted in decreased proliferation of ITDR cells. Further, combined treatment with Flt3-directed TKIs and ZnPP or HO-1 knockdown resulted in increased induction of cell death of ITDR cells. These data provide impetus for direct targeting of HO-1 in TKI resistant ITD+ AML. Together, our data suggest HO-1 as a growth and resistance factor in Flt3-ITD+ AML; therefore, targeting HO-1, or the mechanisms that control its expression, may prove therapeutically valuable. Citation Format: Mary E. Irwin, Joya Chandra. Inhibition of heme oxygenase 1 decreases proliferation and resensitizes TKI-resistant Flt3-ITD-positive AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3591. doi:10.1158/1538-7445.AM2015-3591

Abstract The primary oncogene associated with CML is BCR/ABL which controls proliferative and survival signaling and is a potent inducer of reactive oxygen species (ROS). ROS play both positive and negative roles in proliferation and survival; this dual nature has been exploited by leukemia cells to promote growth, genomic instability, and drug resistance. However, the distinct molecular alterations that occur as a result of BCR/ABL-induced ROS are not well described. BCR/ABL-targeted therapeutics have improved clinical response rates, therefore the number of CML patients living with detectable disease burden is rising. Complete hematologic responses to tyrosine kinase inhibitor (TKI) therapy are seen in ∼10-30% of CML patients, so acquired drug resistance and relapse remain major issues. Thus, novel targets for combined therapeutics are needed. We have shown that early growth response 1 (Egr-1) is a BCR/ABL-dependent, redox-responsive transcription factor that modulates expression of the non-receptor tyrosine kinase Fyn in CML leading to proliferation and survival. Tissue microarray analysis of 26 CML patients (10 chronic phase, 6 accelerated phase, and 10 blast crisis), corroborated by western blotting on independent patient samples, showed that Egr-1 protein expression increased as CML progresses from chronic phase to the more treatment resistant accelerated phase and blast crisis. Egr-1 protein expression was also elevated 2.5 fold in a model of acquired pan-TKI resistance (K562-STI) when compared with parental K562 cells. When Egr-1 was genetically inhibited, proliferation of K562-STI cells decreased by 56%. Egr-1 knockdown was also sufficient to sensitize K562-STI cells to growth inhibition caused by first and second generation BCR/ABL-directed TKI, further implicating Egr-1 in acquired TKI resistance. Egr-1 is well known as a redox-responsive transcription factor, and we found that ROS were elevated in K562-STI vs. K562 cells. While analysis of mitochondrial respiration using a Seahorse Bioanalyzer showed no increase in mitochondrial respiration, spare respiratory capacity, nor proton leak in K562-STI vs. K562, there remained a basal level of oxygen consumption from non-mitochondrial sources in both cell lines. Interestingly, fluorigenic and western blotting assays showed increases in NADPH oxidase (NOX) activity (1.35 fold) and p47phox (2 fold), an essential component of the NOX complex, respectively in K562-STI cells, suggesting NOX as a source of ROS in these cells. To this end, inhibition of the NOX complex with diphenyleneiodonium decreased ROS levels and Egr-1 expression by 50% in K562-STI but not K562 cells. These data suggest that K562-STI have altered regulation of Egr-1 controlled, in part, by NOX. Together, our findings suggest that targeting the transcription factor Egr-1 directly, or through the NOX complex, may be beneficial for improving outcomes for CML patients. Citation Format: Mary E. Irwin, Roxsan Manshouri, Blake Johnson, Hesham M. Amin, Joya Chandra. Targeting Egr-1 is an effective strategy for overcoming kinase inhibitor resistance in CML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2014-960

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic cancer. Thirty percent of AMLs express mutations in the FMS-like tyrosine kinase receptor-3 (FLT3) that render it constitutively active. The internal tandem duplication (ITD) mutation of FLT3 is associated with poor clinical prognosis as it results in aberrant signaling leading to proliferation and survival. As such, tyrosine kinase inhibitors (TKI) directed against FLT3 are being developed. However, as with most TKI therapies, de novo and acquired resistance occurs. We have developed an isogenic cell line model of acquired resistance to FLT3 TKIs by treating BaF3/FLT3-ITD cells (FLT3-ITD) with lestaurtinib over time (FLT3-ITDR cells). Challenge of FLT3-ITDR cells with lestaurtinib showed >3-fold resistance to this TKI as measured by proliferation and propidium iodide sub-diploid analysis. In addition, we tested FLT3-ITDR cells for sensitivity to quizartinib, a more potent and selective inhibitor of FLT3, and found that FLT3-ITDR cells were 7-fold more resistant, suggesting pan-FLT3-TKI resistance. To confirm this resistance was specific for TKI, FLT3-ITDR and FLT3-ITD cells were treated with the histone deactylase inhibitor vorinostat and the anthracycline doxorubicin. Similar sensitivities were noted between the two cell lines to both of these drugs suggesting that resistance of FLT3-ITDR cells to FLT3-directed TKI is specific. Combination therapy is often utilized as a mechanism to overcome clinical resistance. Recently, proteasome inhibitors have been explored as an option for combinatorial therapeutics in leukemia. To determine if the proteasome may be a good target for combination therapy in FLT3-ITDR cells, the proteasomal chymotrypsin-like activity was measured in FLT3-WT, FLT3-ITD, and FLT3-ITDR cells. Chymotrypsin-like proteasome activity was significantly reduced in both FLT3-ITD (41%) and FLT3-ITDR (59%) vs. FLT3-WT cells. This activity stems from activation of the β5 subunit of the proteasome; therefore we performed western blotting with antibodies directed against β5. As seen with chymotrypsin-like activity, FLT3-ITD cells had a significant decrease in β5 protein expression (58%) compared to FLT3-WT. However, despite decreased proteasome activity, FLT3-ITDR had no decrease in β5 protein compared to FLT3-WT cells. One potential explanation for this result is that FLT3-ITDR cells have increased levels of inactive free β5 subunits compared to FLT3-WT. Future studies will examine the implication of these alterations. Despite these changes in proteasome sub-unit regulation and activity, all three cell lines were sensitive to single agent treatment with the reversible FDA-approved proteasome inhibitor bortezomib and its irreversible counterpart marizomib. As such, we will continue to explore the efficacy of proteasome inhibitors in combination therapy for TKI resistant FLT3-ITD-positive AML. Citation Format: Katie Wilson, Mary E. Irwin, Joya Chandra. Proteasomal alterations in a newly created model of FLT3-ITD positive AML with acquired pan-TKI resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1711. doi:10.1158/1538-7445.AM2014-1711

Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants.

Panobinostat, a potent pan-histone deacetylase inhibitor (HDACi) is emerging as a valuable therapeutic option for cancer treatment. Compared with the FDA-approved vorinostat, it displays a more potent and broader spectrum of inhibitory activity (inhibiting all class I, II and IV HDACs) at clinically achievable concentrations. Combinatorial therapies with proteasome inhibitors (PI) have been recently evaluated. Marizomib, a second-generation irreversible PI, exhibits excellent pre-clinical activity against numerous hematologic malignancies. Indeed, published work from our laboratory has shown that marizomib is more effective than the FDA approved PI bortezomib in inhibiting the activity of the proteasome. Additionally, marizomib has been shown to induce higher caspase-8 and reactive oxygen species (ROS) dependent cell death than bortezomib, alone and in combination with HDACi in acute lymphoblastic leukemia (ALL) models. Despite progress in the treatment of acute myeloid leukemia (AML), 40% of patients die from disease recurrence or treatment toxicities; therefore, targeted approaches allowing for low drug doses are needed to increase clinical efficacy. The objectives of this study were to (1) determine if a panobinostat/PI regimen displayed synergy; (2) whether cell death by this combination triggers an unique profile of caspase activation; and (3) assess utility of this combination in a bortezomib refractory setting. Human-derived AML cell lines, ML-1 and AML3, were exposed to increasing concentrations of HDACi, (panobinostat or vorinostat), and PI (bortezomib or marizomib) alone and in combination. Panobinostat had an IC50 within the nanomolar range at 24 hours of treatment; in contrast, an IC50 was not achievable with vorinostat until after 48 hours of treatment. Marizomib was found to have a much lower IC50 and higher DNA fragmentation capacity than bortezomib. Calcusyn software was used to determine synergistic combinations. Synergistic cytotoxicity was observed with the combination treatment of panobinostat with both PIs. No synergy was observed with combinations involving vorinostat. Interestingly, the panobinostat + marizomib combination demonstrated earlier and higher ROS induction. To assess the differences in apoptotic mechanisms between these agents, caspase activities were measured. The combination of panobinostat + marizomib induced an earlier and 2.5x higher induction of caspase-3 activation than the combination of panobinostat + bortezomib. Caspase-8 and caspase-9 dependence, were evaluated using pre-treatment with specific caspase-8 (IETD-fmk) and caspase-9 (LEHD-fmk) inhibitors. Caspase-8 inhibition decreased the cytotoxicity of the panobinostat + marizomib combination compared to control, whereas no difference was observed on cells treated with panobinostat + bortezomib. Western blotting for cleaved caspase-8 corroborated these data. Caspase-9 inhibitors did not significantly protect against DNA fragmentation in any of the combinations. These AML results were consistent with our prior published work using ALL cells, highlighting a role for caspase-8 in sensitivity to synergistic combinations of HDACi and PI. Bortezomib resistance is an emerging problem in the treatment of hematological malignancies. Therefore, RPMI-8226vr10 (bortezomib-resistant multiple myeloma) cells were treated with equimolar doses of either of the HDACi; panobinostat was able to induce higher DNA fragmentation than vorinostat. When panobinostat was combined with either PI, the combination with marizomib caused higher DNA fragmentation than the bortezomib combination. Because caspase-2 has been implicated in panobinostat cytotoxicity in other systems, caspase-2 cleavage was assessed by western blotting assays. At 12 hours of treatment, panobinostat + marizomib caused stronger caspase-2 cleavage than the panobinostat + bortezomib combination, suggesting that marizomib may uniquely augment the caspase-2 activating capacity of panobinostat, providing insight into a potential mechanism of bortezomib resistance. Further experiments will focus on the molecular mechanisms of panobinostat and marizomib combinations and how efficacy could be further optimized. Overall, these data support the use of these novel anticancer agents in hematological malignancies. Disclosures: Orlowski: Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Resverlogix: Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity’s Board of Directors or advisory committees.

Acute myelogenous leukemia (AML) afflicts ∼12,330 new patients per year in the United States. Regrettably, only 25% of patients will survive five years past diagnosis. The most common mutation in AML is internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (Flt3), which renders it constitutively active. Flt3-ITD regulates proliferation and survival, and also increases the production of reactive oxygen species (ROS), which act as secondary messengers for oncogenic signaling. ROS can cause the induction of a number of molecules, however, one protein, heme oxygenase 1 (HO-1), a well-known antioxidant, has been connected to both proliferation and drug resistance of various cancers. We hypothesized that Flt3-ITD-dependent signaling and ROS production increase constitutive expression of HO-1, leading to the activation of antioxidant and anti-apoptotic pathways, resulting in proliferation and drug resistance in AML. Western blotting revealed a two-fold increase of HO-1 protein in Flt3-ITD + cells as compared to Flt3-WT; a four-fold up-regulation of HO-1 mRNA was noted by quantitative real-time PCR suggesting transcriptional control. To determine if this up-regulation was due to the altered redox status of Flt3-ITD + cells, the flavonoid inhibitor diphenylene iodonium (DPI) was used. Consistent with published results implicating the flavonoid protein complex NADPH oxidase (NOX) as a primary source of ROS in these cells, DPI reduced ROS levels as early as two hours post treatment. This ROS reduction coincided with a decrease in HO-1 protein, suggesting that NOX may be involved in HO-1 up-regulation. Our previous results in chronic myeloid leukemia suggest that a Rac1-dependent isoform of NOX controls HO-1 expression, however, in Flt3-ITD + AML, dominant negative inhibition of Rac1 was insufficient to alter HO-1 expression suggesting that either a Rac1-independent NOX isoform is involved or that another flavonoid protein modulates HO-1 expression in this leukemia subtype. Interestingly, when HO-1 expression was knocked down using RNAi, there was a 50% reduction of proliferation and 40% reduction of viability as measured by trypan blue exclusion in Flt3-ITD + AML cells. These data suggest that the function of HO-1 up-regulation is to promote survival and proliferation of Flt3-ITD + AML. Additionally, we have created a model of acquired resistance to lestaurtinib, a Flt3 kinase inhibitor, by treating with increasing doses of the drug over time. Preliminary results suggest that HO-1 is further elevated in these resistant cells as compared to parental Flt3-ITD cells; thus, we will use this model to test the functional role of HO-1 in acquired resistance. Together, our data suggest that HO-1 is a growth and survival factor in Flt3-ITD + AML; therefore, targeting HO-1 or the mechanisms that control its expression may prove therapeutically valuable. Citation Format: Mary E. Irwin, Joya Chandra. The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4124. doi:10.1158/1538-7445.AM2013-4124

Reactive oxygen species (ROS) play both positive and negative roles in the proliferation and survival of a cell. This dual nature has been exploited by leukemia cells to promote growth, survival, and genomic instability-some of the hallmarks of the cancer phenotype. In addition to altered ROS levels, many antioxidants are dysregulated in leukemia cells. Together, the production of ROS and the expression and activity of antioxidant enzymes make up the primary redox control of leukemia cells. By manipulating this system, leukemia cells gain proliferative and survival advantages, even in the face of therapeutic insults. Standard treatment options have improved leukemia patient survival rates in recent years, although relapse and the development of resistance are persistent challenges. Therapies targeting the redox environment show promise for these cases. This review highlights the molecular mechanisms that control the redox milieu of leukemia cells. In particular, ROS production by the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidoreductase, and cytochrome P450 will be addressed. Expression and activation of antioxidant enzymes such as superoxide dismutase, catalase, heme oxygenase, glutathione, thioredoxin, and peroxiredoxin are perturbed in leukemia cells, and the functional consequences of these molecular alterations will be described. Lastly, we delve into how these pathways can be potentially exploited therapeutically to improve treatment regimens and promote better outcomes for leukemia patients. Antioxid. Redox Signal. 18, 1349-1383.

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph(+)ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph(+)ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ~30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph(+)ALL as compared to just 4.8% of Ph(-)ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph(+)ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.

BACKGROUND: Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster regionv-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) oncogene. Recent work has demonstrated that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1expressing cells and that the inhibition of HO-1 in CML leads to reduced cellular growth, suggesting that HO-1 may be a plausible target for therapy. The objective of the current study was to clarify the mechanism of HO-1 overexpression and the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a contributor to this mechanism in CML. METHODS: HO-1 expression was evaluated in bone marrow specimens from patients with CML in various stages of disease, in a transplantation-based model for CML, and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. RESULTS: Specimens from patients with CML in blast crisis displayed higher levels of HO-1 staining than specimens from patients with CML in chronic or accelerated phase. HO-1 up-regulation in BCR-ABL1expressing cells was suppressed by diphenyleneiodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNA interference (RNAi) to Ras-related C3 botulinum toxin substrate 1 (Rac1), a dominant-negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed toward the 47-kd cytosolic subunit of Nox (p47phox) similarly abrogated HO-1 levels. CONCLUSIONS: BCR-ABL1 expression up-regulated HO-1, a survival factor for CML cells. This up-regulation was more pronounced in blast crisis CML relative to early stage disease and was mediated by the NADPH oxidase components Rac1 and p47phox. The expression of p47phox was increased in BCR-ABL1expressing cells. Cancer 2011. (c) 2011 American Cancer Society.

The BCR-ABL1 oncogene is a tyrosine kinase that activates many signaling pathways, resulting in the induction of chronic myeloid leukemia (CML). Kinase inhibitors, such as imatinib, have been developed for the treatment of CML; however, the terminal, blast crisis phase of the disease remains a clinical challenge. Blast crisis CML is difficult to treat due to resistance to tyrosine kinase inhibitors, increased genomic instability and acquired secondary mutations. Our recent studies uncovered a role for Fyn in promoting BCR-ABL1 mediated cell growth and sensitivity to imatinib. Here we demonstrate that Fyn contributes to BCR-ABL1 induced genomic instability, a feature of blast crisis CML. Bone marrow cells and mouse embryonic fibroblasts derived from Fyn knockout mice transduced with BCR-ABL1 display slowed growth and clonogenic potential as compared to Fyn wild-type BCR-ABL1 expressing counterparts. K562 cells overexpressing constitutively active Fyn kinase were larger in size and displayed an accumulation of genomic abnormalities such as chromosomal aberrations and polyploidy. Importantly, loss of Fyn protected mouse embryonic fibroblast cells from increased number of chromosomal aberrations and fragments induced by BCR-ABL1. Together, these results reveal a novel role for Fyn in regulating events required for genomic maintenance and suggest that Fyn kinase activity plays a role in the progression of CML to blast crisis.

Activation of the epidermal growth factor receptor (EGFR) regulates cellular proliferation, survival and migration of breast cancer cells. In particular, EGFR recruits signaling proteins to the cell membrane leading to their phosphorylation and activation. However, EGFR also localizes to other cellular structures, including endosomes, mitochondrion and nuclei. Recently, we demonstrated that lipid raft localization of EGFR in triple-negative breast cancer cell lines promotes EGFR protein-dependent, EGFR kinase-independent activation of Akt. Here, we further define the mechanism by which lipid rafts regulate EGFR signaling to Akt. Specifically, we show that the non-receptor tyrosine kinase c-Src co-localizes and co-associates with EGFR and lipid rafts. Breast cancer cells resistant to treatment with EGFR inhibitors, were also resistant to treatment with Src family kinase (SFK) inhibitors; however, the combination of EGFR and SFK inhibitors synergistically decreases cell viability. We found that this decrease in cell viability observed with EGFR and SFK inhibitor co-treatment correlates with loss of Akt phosphorylation. In addition, we found that in breast cancer cell lines with EGFR and c-Src co-localized to lipid rafts, phospho-inositide-3-kinase (PI3K) was also associated with lipid rafts. Together, the data herein suggest that lipid rafts provide a platform for the interaction of EGFR, c-Src and PI3K, leading to activation of cellular survival signaling in breast cancer cells.

Abstract 1397 The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL. Disclosures: Mills:Glaxosmithkline: Research Funding; Pfizer: Research Funding.