KIT with D816 Mutations Cooperates with CBFB-MYH11 for Leukemogenesis in Mice

Abstract Abstract 867 KIT mutations are the most common secondary mutations in inv(16) AML patients (10–45%), and their presence may suggest poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis and to investigate the underlying mechanism. Here, we transduced bone marrow (BM) cells from wild type (WT) and conditional Cbfb-MYH11 knock-in (Cbfb+/56m; Tg(Mx1-Cre)) mice with retroviral vectors carrying wildtype (WT) or mutant (activation mutations D816V or D816Y) KIT variants. In colony forming assays, KIT (WT or D816 mutants) transduction led to significantly fewer colonies (> 7 fold decrease) from WT BM cells, whereas the Cbfb+/56m; Tg(Mx1-Cre) BM cells were only mildly affected (1.6 fold decrease in colony numbers). Further analysis of transduced BM cells indicated that KIT transduction significantly (p<0.05) increased cell death in transduced Lin− BM cells (both Cbfb+/56m; Tg(Mx1-Cre) and WT), as compared to untransduced Lin− BM cells, which could explain the decreased total colony numbers. Analysis of WT Lin− BM cells transduced with WT KIT and D816 mutants showed similar massive cell death (87% (wt); 87% (D816Y); 94% (D816V)(N=4 for each). On the other hand, in transduced Cbfb+/56m; Tg(Mx1-Cre) Lin− BM cells, the cell death rates were 74.3% (wt), 55.2% (D816Y) and 84.5% (D816V)(N=4 for each). This difference in the level of cell death could explain the differential effects of KIT on colony formation from transduced Cbfb+/56m; Tg(Mx1-Cre) BM cells vs. WT BM cells. These results also suggest that BM cells expressing Cbfb-MYH11 are more resistant to the toxic effects of KIT than WT BM cells. Moreover, more mixed-lineage (CFU-GEMM) and fewer erythroid (BFU-E) colonies were obtained from Cbfb+/56m; Tg(Mx1-Cre) BM cells transduced with D816V/Y KIT than those transduced with WT KIT, suggesting differentiation defects in early myeloid and erythroid progenitors were induced by the mutant KIT. We then transplanted the transduced BM cells and found that 60% and 80% of mice transplanted with Cbfb+/56m; Tg(Mx1-Cre) BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, while none of the control mice did. Results from limiting dilution transplantations using multiple donor leukemia cells (N=3) showed that mice transplanted with as little as 10 cells died from leukemia within two month, while mice transplanted with 106Cbfb+/56m; Tg(Mx1-Cre) leukemia cells died around two months. We also found a significant increase of mitotic cells in Cbfb+/56m; Tg(Mx1-Cre) leukemic spleen cells that carried the KIT mutations. These data indicate that the KIT D816 mutations not only facilitate the transformation of Cbfb+/56m; Tg(Mx1-Cre) BM cells to leukemia cells, but also help maintain these leukemia cells with higher leukemia initiating cells and proliferation. We next explored the response of this aggressive leukemia to a novel small molecule, the kinase inhibitor PKC412, and found that Cbfb+/56m; Tg(Mx1-Cre) leukemia cells carrying KIT D816 mutations were sensitive to this kinase inhibitor, with significantly less survival than leukemia cells without mutant KIT and cells treated by vehicle only after overnight treatment. Signaling pathway analysis of these leukemia cells suggested that Stat3 and P44/42 MAPK signaling, which has been reported to be activated in cancer cells and is involved in cell proliferation, might be imported for these leukemia cells. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis and show that acute myeloid leukemia cells carrying the inv(16) fusion gene and an activating KIT mutation respond to the small molecule PKC412. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

C-KIT with D816Y/V Mutations Cooperate with CBFB-MYH11 to Accelerate Leukemogenesis in Mice.

Blood ◽

10.1182/blood.v114.22.274.274 ◽

2009 ◽

Vol 114 (22) ◽

pp. 274-274 ◽

Cited By ~ 1

Author(s):

Ling Zhao ◽

J. Joseph Melenhorst ◽

Lemlem Alemu ◽

Martha Kirby ◽

Stacie M Anderson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Retroviral Vectors ◽

Serine Phosphorylation ◽

Leukemia Cells ◽

Wild Type ◽

Average Life Span ◽

Kit Mutation ◽

Clinical Prognosis ◽

Chemical Screening ◽

Type C

Abstract Abstract 274 CBF leukemia is a group of acute myeloid leukemia (AML) caused by the fusion genes involving core binding factors (CBFs), which include RUNX1 and CBFB. C-KIT mutations are common in CBF leukemias. For example, 10–45% of AML cases with inv(16), which generates a fusion gene between CBFB and MYH11, carry activating mutations in c-KIT. In general, the presence of c-KIT mutations in inv(16) AML suggests poor clinical prognosis. Therefore, it is important to verify that c-KIT mutations cooperate with CBFB-MYH11, and to investigate the underlying mechanism during leukemogenesis. Here, we transduced bone marrow (BM) cells from conditional Cbfb-MYH11 knockin mice with retroviral vectors carrying c-KIT mutations,D816V and D816Y (located in the kinase domain of c-KIT and can cause constitutive activation of c-KIT), which are common in inv(16) AML. Wild type c-KIT and empty retroviral vectors were used as controls. The transduced BM cells were then transplanted to sub-lethally irradiated recipient mice. In 2–5 months, 60% of mice carrying D816Y and 50% of mice carrying D816V c-KIT mutations developed leukemia, while none of the mice transduced with the control vectors developed leukemia up to one year after transplantation. In addition, BM cells from wildtype mice transduced with the c-KIT mutant vectors did not induce leukemia >5 months after transplantation. Analysis of signaling pathways revealed that Stat3-serine phosphorylation and P44/42 MAPK pathways, but not AKT and Stat5 pathways, were activated (phosphorylated) in the leukemia cells from the Cbfb-MYH11 knockin mice expressing mutated c-KIT. In vitro colony forming assays indicated that Cbfb-MYH11 BM cells expressing c-KIT D816V/Y mutants produced similar number of colonies as Cbfb-MYH11 BM cells transduced with wild type c-KIT control vector. However, they had increased CFU-G and CFU-GEMM and decreased CFU-E colonies, suggesting that the c-KIT mutants drove Cbfb-MYH11 BM cells towards an earlier progenitor state. Secondary transplantation with 1×106 leukemia cells from donors with c-KIT mutations showed an average life span of three weeks compared to six weeks from donors without c-KIT mutation. Further limiting dilution transplantation with 100 leukemia cells from one donor with c-KIT mutation led to lethal leukemia in 4 out of 5 mice 2.5 months after transplantation, indicating a very high frequency of leukemia initiating cells in this sample. These data suggest a strong cooperation between the mutant c-KIT and Cbfb-MYH11 for leukemogenesis in mice. This mouse model can also serve as a clinically relevant model for small chemical screening and the development of novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Erythropoietin Regulates GATA-1 Function and Erythroid Differentiation by a PKD-HDAC5 Signal Transduction Pathway

Blood ◽

10.1182/blood.v116.21.85.85 ◽

2010 ◽

Vol 116 (21) ◽

pp. 85-85

Author(s):

Lorrie L. Delehanty ◽

Adam N. Goldfarb

Keyword(s):

Cell Death ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Class Ii ◽

Dependent Manner ◽

Wild Type ◽

Erythroid Progenitors ◽

Nadir Point ◽

Shrna Knockdown

Abstract Abstract 85 How erythropoietin (Epo) signaling promotes erythroid differentiation remains unclear. Epo is known to regulate the function of the master regulator of erythroid gene transcription, GATA-1. Using murine proerythroblasts engineered with a conditional GATA-1-ER fusion, G1ER cells, Gregory et al. showed that GATA-1 induction of erythroid differentiation required signaling by Epo; in cultures lacking Epo, activation of GATA-1 caused cell death without differentiation (Blood 94:87-96, 1999). Although several models have been proposed for Epo regulation of GATA-1, no mechanism has been established. While analyzing PKC isozymes regulated by Epo and iron in primary human erythroblasts, our lab identified PKCμ , aka PKD, as activated in a dosage-dependent manner by Epo. Subsequent studies in Epo-starved G1ER cells, and in primary human progenitors, confirmed direct Epo induction of PKD phosphorylation on Ser744/748. A major PKD pathway involves its phosphorylation of class II HDACs (4, 5, 7 and 9), leading to release of HDAC-bound transcription factors (e.g. MEF2) from tonic repression. Watamoto et al. previously identified GATA-1 as a class II HDAC regulated factor, displaying physical and functional interaction with HDAC5 (Oncogene 22:9176-9184, 2003). Accordingly, we employed G1ER cells to determine whether Epo signaling to GATA-1 involved the PKD-HDAC pathway. In G1ER cells cultured in stem cell factor (SCF) minus Epo, endogenous HDAC5, but not HDAC4, co-immunoprecipitated with GATA-1. Epo stimulation induced dissociation of the HDAC5-GATA-1 complex, without affecting levels of either factor. The function of erythroid PKD-HDAC signaling was addressed by pharmacologic and shRNA approaches. Initial experiments tested whether inhibition of HDAC activity could substitute for Epo signaling in G1ER cells undergoing estradiol activation of GATA-1-ER. As described, GATA-1 activation in cells grown in SCF without Epo caused >90% cell death with minimal hemoglobinization after 48 hours. Addition of the HDAC inhibitor SAHA completely rescued viability and partially restored hemoglobinization of cells undergoing GATA-1 activation in the absence of Epo. Along similar lines, shRNA knockdown of HDAC5, but not HDAC4, significantly enhanced viability and hemoglobinization of G1ER cells undergoing GATA-1 activation under limiting Epo concentrations (0.05 U/ml). Conversely, shRNA knockdown of PKD3 impaired hemoglobinization of cells undergoing GATA-1 activation in the presence of Epo. Further implicating PKD in erythropoiesis, differentiation of G1ER cells was blocked by the kinase inhibitor Gö6976, which targets PKC and PKD, but not by the related compound Gö6983, which targets only PKC. Similar results were obtained with primary human progenitors, in which the PKD inhibitor Gö6976 but not the classical PKC inhibitor Gö6983 inhibited erythroid differentiation. In vivo roles of PKD-HDAC signaling in erythropoiesis were addressed by studying HDAC5-/- mice. In contrast to wild type counterparts, adult HDAC5-/- mice showed elevated steady state hematocrits (56.2 ± 0.8 vs 39.3 ± 1.4, P < 0.0001), resulting from increased MCVs (68.8 ± 0.7 vs 48.2 ± 0.1, P < 0.0001). In response to PHZ-induced hemolytic anemia, HDAC5-/- mice showed higher nadir RBC counts (5.0 ± 0.3 vs 3.2 ± 0.2, P < 0.001) and displayed an unexpected increase in Hb levels at the nadir point (15.9 ± 0.8 vs 8.6 ± 0.5, P < 0.0001). Spleens and livers obtained post recovery showed increased iron deposition in HDAC5-/- mice, consistent with increased net red cell turnover. To determine whether the observed abnormalities were cell-intrinsic, we sorted CD71++ Ter119- erythroid progenitors from the marrows of adult HDAC5-/- and wild type mice. The HDAC5-/- progenitors, but not wild type counterparts, displayed erythroid differentiation, manifested by Ter119 upregulation, in medium with no or low Epo (0.01-0.05 U/ml). Our data thus implicate PKD-HDAC signaling in Epo regulation of GATA-1 function and thereby provide a mechanistic basis for an instructional function of the Epo receptor. In addition, activation of PKD in non-erythroid cells could potentially explain some of the poorly-understood clinical complications of Epo therapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412

Cancer Cell ◽

10.1016/s1535-6108(02)00069-7 ◽

2002 ◽

Vol 1 (5) ◽

pp. 433-443 ◽

Cited By ~ 415

Author(s):

Ellen Weisberg ◽

Christina Boulton ◽

Louise M Kelly ◽

Paul Manley ◽

Doriano Fabbro ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Small Molecule ◽

Kinase Inhibitor ◽

Leukemia Cells

Download Full-text

Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Blood ◽

10.1182/blood.v116.21.1809.1809 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1809-1809

Author(s):

Hisashi Harada ◽

Nastaran Heidari ◽

Mark Hicks

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Small Molecule ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Annexin V ◽

Glucocorticoid Resistance ◽

Down Regulation

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CXCL12 Enhances Survival of Chronic Lymphocytic Leukemia Cells Via a RAF-Dependent Pathway That Can Be Inhibited by Sorafenib

Blood ◽

10.1182/blood.v116.21.2445.2445 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2445-2445

Author(s):

Davorka Messmer ◽

Jessie-F Fecteau ◽

Morgan O'Hayre ◽

Tracy Handel ◽

Thomas J. Kipps

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Small Molecule ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Growth Factor Receptor ◽

Lymphocytic Leukemia ◽

Increased Risk ◽

Dependent Pathway

Abstract Abstract 2445 The cellular microenvironment is critical for the survival of Chronic Lymphocytic Leukemia (CLL) cells. CLL cells die rapidly in vitro unless they receive survival signals from stromal cells or “nurse-like” cells (NLCs). CLL cell survival is in part mediated by the stromal cell-derived factor-1 (SDF-1alpha, designated as CXCL12), which is expressed by NLCs. CXCL12 is a highly conserved chemokine that can promote CLL-cell survival through its receptor CXCR4. Prior studies showed that treatment of CLL cells with CXCL12 induced activation of Extracellular Signal-Regulated Kinase (ERK). In this study, we examined CXCL12 signaling in CLL cells to characterize the mechanism (s) accounting for its ability to enhance CLL-cell survival. For this we examined CLL cells with high- or low- level expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that is expressed by CLL cells of patients who have an increased risk for early disease progression and short survival. We found that CXCL12 induced a robust intracellular Ca2+ flux in ZAP-70+ CLL cells but only modest-to-poor Ca2+ flux in ZAP-70-negative CLL cells. Furthermore, ZAP-70+ CLL cells (n=10) responded to CXCL12 stimulation with increased and prolonged phosphorylation of ERK and MEK compared to ZAP-70-negative CLL cells (n=9). To investigate the underlying mechanism for MEK activation in ZAP-70+ CLL, we used small molecule inhibitors and found that CXCL12-induced phosphorylation of ERK and MEK could be blocked by sorafenib, a small molecule inhibitor of RAF. The role of RAF was further supported using KG5, a kinase inhibitor of RAF signaling through B-RAF and C-RAF in addition to platelet-derived-growth-factor-receptor (PDGFR) alpha and beta, Flt3, and Kit. As a control, we used a kinase inhibitor that targets all of these kinases except B- and C-RAF (KG1) and found it could not inhibit MEK activation. The involvement of Raf was further substantiated using GW5074, an inhibitor of B-RAF and C-RAF. Both KG5 and GW5074 could inhibit CXCL12-induced MEK activation in ZAP-70+ CLL samples. CXCL12-induced activation of MEK/ERK was not affected by sunitinib, an inhibitor of non-RAF kinases that also are inhibited by sorafenib, including VEGFR, PDGFR, Flt3, and c-Kit. Sorafenib not only inhibited MEK/ERK activation but also caused apoptosis of CLL cells whereby ZAP-70+ CLL cells showed incresed sensitivity to lower doses of sorafenib. Consistent with these results we found that ZAP-70+ CLL cells had a greater responsiveness to CXCL12 for survival in vitro than did ZAP-70-negative CLL cells. We conclude that CXCL12 can enhance survival particularly of ZAP-70+ CLL cells via a RAF dependent pathway, which can be targeted by the kinase inhibitor sorafenib. As such, sorafenib might be effective in blocking the protective influence of the microenvironment on CLL cells, suggesting that this drug could have activity in the treatment of patients with this disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Flt3/ITD Confers Resistance to FLT3 Inhibitor AC220 By up-Regulating Runx1

Blood ◽

10.1182/blood.v124.21.2223.2223 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2223-2223

Author(s):

Tomohiro Hirade ◽

Mariko Abe ◽

Chie Onishi ◽

Seiji Yamaguchi ◽

Seiji Fukuda

Keyword(s):

Tyrosine Kinase ◽

Prolonged Exposure ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Specific Inhibitor ◽

Hematopoietic Stem ◽

Runx1 Expression ◽

Cells Cultured ◽

Resistant Cells

Abstract FMS-like tyrosine kinase 3 (FLT3) is a membrane type tyrosine kinase and has important roles for the proliferation and differentiation of the hematopoietic cells. The Internal Tandem Duplications of FLT3 (FLT3/ITD) is detected in approximately 30 % of patients with acute myeloid leukemia (AML) and the prognoses of FLT3/ITD+ AML are very poor. While a number of inhibitors targeting FLT3 tyrosine kinase have been developed, few drugs are effective for the FLT3/ITD+ AML because of emergence of resistant cells against the drugs. Recently, AC220 (Quizartinib), a second generation class III tyrosine kinase inhibitor (TKI) for FLT3/ITD+ AML was developed and used in clinical trials. Although AC220 is a more potent and specific inhibitor for FLT3/ITD compared to the other TKIs, report demonstrates that prolonged exposure to AC220 can generate resistant clones to AC220 in FLT3/ITD+ cells (Smith et al. Nature 2012). These findings underscore the need to develop additional therapeutic strategies to overcome the resistance of FLT3/ITD+ AML to TKIs. However, the mechanism responsible for drug resistance of FLT3/ITD+ AML cells remains to be investigated. We previously reported that mRNA expression of RUNX1, a core-binding transcription factor that regulates the differentiation and proliferation of hematopoietic stem cells, is significantly higher in FLT3/ITD+AML cells compared to FLT3/ITD-AML cells (Hirade et al. ASH 2013). Although loss of RUNX1 function (i.e. RUNX1/ETO fusion gene) contributes to the development of AML, RUNX1 also promotes survival of AML cells (Goyama et al. JCI 2013) and can function as an oncogene in cancer cells (Kilbey et al. Cancer Research 2010). These findings lead us hypothesize that RUNX1 may confer resistance of AML cells to TKIs. In the present study, we investigated if Runx1 is involved in the refractory phenotype of Flt3/ITD+cells to AC220. Transduction of Flt3/ITD into IL3-dependent mouse 32D cells allowed the cells proliferate in a growth factor independent fashion, concomitant with up-regulation of Runx1 mRNA level, similar to the patients’ samples with FLT3/ITD+AML. Silencing Runx1 expression using shRNA resulted in 70% reduction of Flt3/ITD+32D cells that proliferated in the absence of growth factors. Similarly, incubating the Flt3/ITD+32D cells with 0.5nM AC220 inhibited their factor independent proliferation by 95%, which was further accentuated up to 99% by the combination with shRNA mediated silencing of Runx1. Although the number of factor independent Flt3/ITD+32D cells cultured in the presence of 2nM AC220 rapidly declined within 96 hours, the residual cells subsequently re-proliferate within 14 days and became no longer sensitive to AC220. Surprisingly, the expression of Runx1 mRNA in the resistant cells to AC220 was 5.0±0.2 fold higher (P<0.05) compared to control Flt3/ITD+32D cells sensitive to AC220. Silencing Runx1 using shRNA abrogated the proliferation of AC220-resistant Flt3/ITD+32D cells cultured in the presence of 2nM AC220, leading to 99.5% reduction in the viable cells. Our data indicates that knocking down Runx1 expression enhances the cytotoxic effect of AC220 on Flt3/ITD+32D cells and that Runx1 expression is significantly up-regulated by the AC220 resistance cells. Moreover, Runx1 knockdown recovered the cytotoxicity of AC220 in the refractory Flt3/ITD+32D cells, demonstrating that Flt3/ITD confers resistance to AC220 by up-regulating the expression of Runx1. These findings demonstrate that antagonizing RUNX1 may represent potential therapeutic strategy in the patients with FLT3/ITD+ AML that become refractory to AC220. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Identification of the Histone Deacetylase Inhibitor (HDACi), AR-42, as a Novel Anti-Leukemia Stem Cell Agent in Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v114.22.2070.2070 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2070-2070

Author(s):

Duane Hassane ◽

Marlene Balys ◽

Guido Marcucci ◽

John C. Byrd ◽

Craig T. Jordan ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Stem Cell ◽

Colony Formation ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Expression Pattern ◽

Parp Cleavage ◽

Gene Expression Patterns ◽

Erythroid Progenitors

Abstract Abstract 2070 Poster Board II-47 Despite recent progress, the majority of AML patients are refractory or relapse following standard cytarabine/anthracycline-based treatments. Studies have demonstrated that leukemia stem cells (LSCs) are able to initiate and perpetuate AML and are reported to be resistant to standard chemotherapeutics. Therefore, it has been hypothesized that these cells contribute to determination of the poor outcome of AML patients. The discovery of novel agents that directly target LSCs may potentially improve these disease outcomes. Previous studies have demonstrated the utility of screening public microarray data for new gene expression patterns mimicking the gene expression patterns of known anti-LSC compounds. Indeed, in screening the NCBI Gene Expression Omnibus (GEO), we found that compounds mimicking the gene expression pattern of a known anti-LSC compound, parthenolide (PTL), could impair the leukemogenic activity of phenotypically- and functionally-defined LSCs. Using this approach, we have reported two novel PTL-like compounds: celastrol and 4-hydroxy-2-nonenal. However, as investigators continue to deposit new data into GEO, we have re-interrogated the GEO database to identify additional compounds active against LSCs . One of our most recent “hits” was the HDACi, AR-42. This compound represents a novel class of HDACis that are structurally similar to phenylbutyrate, but with improved HDACi activity within sub-micromolar concentrations. To validate our screening results, we then tested the efficacy of AR-42 in AML cell lines and primary human AML samples evaluating bulk, progenitor/stem cell populations. Indeed, as predicted by the gene expression-based screen, exposure to 1 microM AR-42 produced greater than 50% cell death at 24 hours post-treatment in phenotypically defined AML stem/progenitor cells. Next, we tested the ability of AR-42 to impair the growth of AML LSC-derived colonies in methylcellulose media. 1 microM AR-42 significantly impaired the formation of AML colonies compared to relative to untreated controls (5% colony formation in AR-42-treated vs untreated; p < 0.05). Importantly, impairment of colony formation of normal myeloid and erythroid progenitors was markedly less pronounced than that of leukemic progenitors, suggesting a preferential activity of this compound on leukemogenic cells. Surprisingly, we observed that AR-42 mechanism of induction of cell death differs from other anti-LSC compounds hitherto identified (parthenolide, celastrol, 4-hydroxy-2-nonenal), which involved NFkappaB inhibition coupled to induction of oxidative stress. Interesting, we found that AR-42 failed to induce strong NFkappaB inhibition and, unlike parthenolide, the AR-42-dependent leukemia-specific death mechanism involved activation of the caspase-3, caspase-8 and PARP cleavage Altogether, these findings indicate the utility of gene expression-based screens to identify novel compounds that are active on LSC, including AR-42 which is being tested in patients with AML and other hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting Phosphotyrosine-315 In RAD51 Recombinase to Prevent BCR-ABL1 - Mediated Unfaithful Homeologous Recombination Repair.

Blood ◽

10.1182/blood.v116.21.1190.1190 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1190-1190

Author(s):

Artur Slupianek ◽

Yashodhara Dasgupta ◽

Shu-yue Ren ◽

Kimberly Cramer ◽

Tomasz Skorski

Keyword(s):

Chromosomal Aberrations ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Chronic Phase ◽

Sh3 Domain ◽

Leukemia Cells ◽

Dsb Repair ◽

Direct Role ◽

Recombination Repair ◽

Homeologous Recombination

Abstract Abstract 1190 Background: CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast phase (CML-BP) and cell lines transformed by non-mutated BCR-ABL1 kinase or tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) (Nowicki et al., Blood, 2006; Cramer et al., Cancer Res., 2008). DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS and radiation induced DSBs generated chromosomal aberrations in BCR-ABL1-positive leukemia cells (Koptyra et al., Leukemia, 2008), which may contribute to the malignant progression to CML-BP. We also showed that homologous recombination repair (HomoRR) driven by RAD51 recombinase is one of the major DSB repair mechanisms, which is stimulated by BCR-ABL1 (Slupianek et al., Molecular Cell, 2001). Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. Here we investigated if BCR-ABL1 can modulate RAD51 recombinase to corrupt the fidelity of recombination and if this process can be targeted to prevent genomic instability in leukemia cells. Result: To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease–inducible DSB site and a repair template displaying 1% sequence divergence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells, BCR-ABL1-32Dcl3 cells and BCR-ABL1(T315I)-32Dcl3 cells. BCR-ABL1 and BCR-ABL1(T315I) kinase caused about a 3-fold increase in HomeoRR activity implicating its role in the accumulation of chromosomal aberrations in CML cells. The magnitude of HomeoRR stimulation depended on BCR-ABL1 expression levels. RAD51 recombinase, a key regulator of recombination repair, forms a complex with BCR-ABL1 which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain of BCR-ABL1, but does not depend on its kinase activity. In fact, BCR-ABL1(K1172R) kinase-dead mutant formed complexes more abundantly with RAD51 than the kinase-active BCR-ABL1. BCR-ABL1-RAD51 complex formation resulted in direct phosphorylation of RAD51 on Y315 [RAD51(phosphoY315)], which is located in the vicinity of PP motifs in the C-terminal portion of RAD51. Phosphorylation-less C-terminal RAD51(Y315F) mutant formed a stronger complex with BCR-ABL1 than the wild-type form. Altogether, it appears that RAD51 PP bind to the SH3 domain of BCR-ABL1 kinase followed by quick phosphorylation of RAD51 on Y315 and disassembly of RAD51(phosphoY315) from the complex. Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. In fact RAD51(phosphoY315) was found in the nuclei of BCR-ABL1 leukemia cells, but not of parental cells and formed numerous foci on DSBs. Phosphorylation-less RAD51(Y315F) mutant abrogated foci formation and inhibited HomeoRR. Thus, BCR-ABL1 – RAD51(phosphoY315) pathway appears to promote unfaithful HomeoRR and chromosomal instability. To test if RAD51(phosphoY315) can be targeted to prevent unfaithful HomeoRR in leukemia cells, a peptide aptamer strategy was applied. Aptamer mimicking the RAD51(phosphoY315) fragment, but not that with the Y315F phosphorylation-less substitution inhibited RAD51 foci formation and HomeoRR activity in BCR-ABL1 leukemia cells. Conclusion: In summary, the BCR-ABL1-RAD51 axis may promote accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR-ABL1 kinase. We hypothesize that targeting BCR-ABL1-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BP progression. Furthermore, RAD51 and recombination is also affected by other oncogenic tyrosine kinases (OTKs) such as TEL-ABL1, TEL-PDGFR, ZNF198-FGFR1, TEL-JAK2, JAK2[V617F], NPM-ALK, IGF-1R, EGFR and FLT3-ITD suggesting that abrogation of OTK-mediated aberrant modulation of RAD51 may inhibit genetic instability and tumor progression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Small Molecule Inhibitors of HER2 Inhibit Proliferative Signaling and Promote Apoptosis in Ph+ ALL

Blood ◽

10.1182/blood.v118.21.1397.1397 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1397-1397

Author(s):

Mary E Irwin ◽

Laura Nelson ◽

Janice M Santiago-O'Farrill ◽

Claudia P Miller ◽

Doris R. Siwak ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Small Molecule ◽

Tyrosine Kinases ◽

Research Funding ◽

Receptor Tyrosine Kinases ◽

Kinase Inhibitor ◽

Her2 Receptor ◽

Small Molecule Kinase Inhibitor ◽

Her2 Signaling

Abstract 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.

Download Full-text