Mary E. Irwin

Redox Control of Leukemia: From Molecular Mechanisms to Therapeutic Opportunities

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.

Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

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.

Expression and Activity of Fyn Mediate Proliferation and Blastic Features of Chronic Myelogenous Leukemia

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.

Inhibition of the NADPH oxidase regulates heme oxygenase 1 expression in chronic myeloid leukemia

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐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‐ABL1–expressing 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.

Abstract 960: Targeting Egr-1 is an effective strategy for overcoming kinase inhibitor resistance in CML

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 1711: Proteasomal alterations in a newly created model of FLT3-ITD positive AML with acquired pan-TKI resistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract 4124: The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML.

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

Inhibition of the NADPH oxidase regulates heme oxygenase 1 expression in chronic myeloid leukemia: Nox2 Influences HO-1 Expression in CML

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐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‐ABL1–expressing 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.

Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐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‐ABL1–expressing 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.