A Recombinant Human Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Shows Selective Cytotoxicity against Myeloid Leukemia Cells and CD123-Positive Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1728-1728
Author(s):  
Varghese Palath ◽  
Rohini Vekhande ◽  
Andreia Lee ◽  
Jason Williams ◽  
Ling Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Surface Expression ◽  
Leukemia Cells ◽  
Human Antibody ◽  
Primary Cells ◽  
Leukemic Stem Cells ◽  
Adcc Activity

Abstract Abstract 1728 Poster Board I-754 Members of the Eph family of receptor tyrosine kinases play important roles in embryonic development but have restricted tissue distribution and activity in adult tissues. The EphA3 receptor is an oncofetal antigen expressed at high levels on the surface of several solid tumor types and certain leukemias. KaloBios is in pre-clinical development with KB004, a high-affinity recombinant human antibody, derived from a monoclonal anti-EphA3 antibody by Antibody HumaneeringTM Technology. KB004 binds EphA3 and stimulates apoptosis in primary cells from myeloid leukemia patients. The cell-surface expression of EphA3 was analyzed by flow cytometry on primary cells from chronic myeloid leukemia (CML) [n=10], acute myeloid leukemia (AML) of various sub-types [n=29], and myelodysplastic syndromes (MDS) [n=7]. EphA3 surface expression was detected in at least 50% of patient samples from each of the diseases analyzed. CD123-positive leukemia stem cells (CD34+ CD38- CD123+) also displayed surface expression of EphA3 whereas normal bone marrow CD34+ stem/ progenitor cells lacked detectable EphA3. KB004 was shown to stimulate apoptosis in EphA3+ primary leukemia cells, including CD123+ leukemic stem cells with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. Cross-linking of the antibody was not required for induction of apoptosis. KB004 also inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony formation. In addition to its direct pro-apoptotic effect, KB004 induced potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3-positive cells, mediated by CD16-expressing effector cells. CD16-dependent ADCC activity was further enhanced by expression of the antibody in CHO cells that lack a1,6 fucosyl transferase, generating afucosylated antibody with higher affinity for CD16a. The data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies in addition to solid tumors. In particular, the selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells that merits clinical investigation. Disclosures No relevant conflicts of interest to declare.

A Recombinant Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Against Various Hematologic Malignancies Shows Selective Inhibition of Colony Formation From Long-Term Culture-Initiating Cells (LTC-ICs) In Primary Leukemia Samples

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2897-2897
Author(s):  
Varghese Palath ◽  
Rohini Vekhande ◽  
Mark Baer ◽  
Wendy Ching ◽  
Nenad Tomasevic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Recombinant Antibody ◽  
Surface Expression ◽  
Hematologic Malignancies ◽  
Adcc Activity ◽  
High Affinity

Abstract Abstract 2897 EphA3 is a receptor tyrosine kinase expressed at high level on the surface of some solid tumors and certain leukemias. KB004 is a high-affinity non-fucosylated, recombinant antibody developed by KaloBios derived from a mouse monoclonal anti-EphA3 antibody using Antibody Humaneering™ Technology. The antibody binds EphA3 from human and cynomolgus macaque with equivalent affinity (610 pM for human and 570 pM for cynomolgus as determined by surface plasmon resonance (spr) analysis) making the latter a relevant toxicology species. KB004 has a direct pro-apoptotic effect on EphA3-positive cells and a induces potent antibody-dependent cellular cytotoxicity (ADCC) activity mediated by CD16-expressing effector cells. The non-fucosylated antibody has enhanced affinity for both human and cynomolgus CD16a. The affinity of KB004 for cynomolgus CD16a (7.2 nM, determined by spr analysis) was comparable to the affinity for the high-affinity variant of CD16a from humans (6.6 nM), indicating that KB004 has similar potential for ADCC in cynomolgus macaques. We first demonstrated cell-surface expression of EphA3 by flow cytometry in approximately 50% of primary human leukemia cells. Here we extend the analysis to multiple myeloma (MM) in which 3/5 patient samples show EphA3 expression. EphA3 expression was confirmed in a panel of chronic myeloid leukemia (CML) samples [n=14] and in acute myeloid leukemia (AML) [n=40] in which EphA3+ cells were identified in each French American British (FAB) classification sub-type except M0, for which only two samples were studied. CD123-positive leukemia stem cells (CD34+ CD38− CD123+) also displayed surface expression of EphA3. KB004 induced apoptosis in EphA3+ primary cells from each of the diseases analyzed, including imatinib-sensitive and resistant CML, with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. KB004 inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony recovery and also showed dose-dependent inhibition of long-term culture-initiating cells (LTC-IC) from AML stem cells. EphA3 expression was not detected on primary human CD34+ bone marrow cells from healthy individuals. These data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies. The selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of MPL in Leukemia Stem Cells and Its Role in Stemness Maintainance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1723-1723 ◽  
Author(s):  
Huan Li ◽  
Qing Rao ◽  
Pei Yu ◽  
Shuying Chen ◽  
Zheng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Colony Formation ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Clonogenic Potential

Abstract Osteoblast cells play an important role in bone marrow niche. The interaction between osteoblast and leukemia cells promotes leukemia development, which is mediated by some cytokines including TPO. It has become evident that TPO-MPL signaling is essential for the quiescence and self-renewal of hematopoietic stem cells, however, its expression pattern and the role in leukemia stem cells have not been reported. This study was aimed to determine the expression of MPL in acute myeloid leukemia (AML) and investigate the role of MPL in the leukemia stem cells' quiescence, drug resistance and self renewal. The expression levels of CD34, CD38 and MPL were detected by flow cytometry in bone marrow cells from 57 newly diagnoses AML patients. The correlation between MPL and CD34, CD38 expression in AML patients were analyzed. The results showed that expression of MPL in AML patients was higher significantly than that in 13 normal donors (P<0.05). Expression of MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P<0.01). MPL was higher expressed in CD34+ cells than that in CD34- cells significantly (P<0.0001). We also detected the expression of MPL in different populations of leukemia cells in AML1-ETO9a mouse leukemia model established in our lab. We found that the ratios of MPL positive cells in Lin-c-kit+ and Lin-c-kit+sca-1+ populations were significantly higher than that in total leukemia cells. In addition, in chemotherapy treated AML1-ETO9a mice, the proportion of Lin-c-kit+MPL+ leukemia cells were increased 23.5 folds than that in untreated leukemia mice, which indicates that MPL+Lin-c-kit+ LSCs population could be enriched by chemotherapy. Furthermore, MPL+ and MPL- cells in Lin-c-kit+ leukemia population were sorted by flow cytometry and the colony formation and quiescence state were determined. The results showed that MPL+Lin-c-kit+ cells produced significantly more colonies in the second round of colony formation (p<0.05) than MPL-Lin-c-kit+ cells. The G0 phase accumulation of MPL+Lin-c-kit+ cells was significantly higher than that of MPL-Lin-c-kit+ cells (p<0.01). Above results indicate that MPL+ leukemia cells display more clonogenic potential and maintain quiescence. These data demonstrate that as a receptor of TPO, MPL is highly expressed in leukemia stem cells and MPL positive leukemia stem cells could be enriched by chemotherapy. MPL positive leukemia stem cells exhibit more clonogenic potential, quiescence and drug resistance. It suggests TPO-MPL mediated interaction of osteoblast and leukemia cells take a role in the stemness of leukemia stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5369-5369
Author(s):  
Seiichi Okabe ◽  
Yuko Tanaka ◽  
Mitsuru Moriyama ◽  
Akihiko Gotoh
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Transfected Cells ◽  
Resistant Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

Inhibitory Effects of Homoharringtonine on Leukemic Stem Cells and BCR-ABL Induced Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2912-2912 ◽  
Author(s):  
Yaoyu Chen ◽  
Yiguo Hu ◽  
Shawnya Michaels ◽  
Dennis Brown ◽  
Shaoguang Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Leukemic Stem Cells ◽  
Lymphoid Leukemia

Abstract The Abl tyrosine kinase inhibitors (TKIs) imatinib mesylate (IM) and dasatinib, targeting BCR-ABL for the treatment of Philadelphia-positive (Ph+) leukemia including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL), have produced impressive results in terms of therapeutic outcome and safety for patients. However, clinical resistance to these TKIs likely at the level of leukemic stem cell negates curative results in Ph+ leukemia. At present, an anti-stem cell strategy has not been developed for treating these leukemia patients. Homoharringtonine (HHT) (omacetaxine mepesuccinate - USAN/INN designation) has shown significant clinical activity in CML in combination with IM or alone for patients failing IM. However, little is known about whether HHT has an inhibitory effect on leukemic stem cells. The purpose of this study is to determine whether HHT inhibits BCR-ABL-expressing leukemic stem cells (Lin-c-Kit+Sca-1+) that we identified previously (Hu et al. Proc Natl Acad Sci USA 103(45):16870–16875, 2007) and to evaluate therapeutic effects of HHT on CML and B-ALL in mice. We find that in our in vitro stem cell assay, greater than 90% of leukemic stem cells were killed after being treating with HHT (12.5, 25, and 50 nM) for 6 days, and in contrast, greater than 75% or 92% of leukemic stem cells survived the treatment with dasatinib (100 nM) or imatinib (2 mM). We next treated CML mice with HHT (0.5 mg/kg, i.p., once a day). 4 days after the treatment, FACS analysis detected only 2% GFP+Gr–1+ myeloid leukemia cells in peripheral blood of HHT -treated CML mice and in contrast, 41% GFP+Gr–1+ myeloid leukemia cells in placebo-treated mice. We also treated mice with BCR-ABL induced B-ALL with HHT, and found that only 0.78% GFP+B220+ lymphoid leukemia cells were detected in peripheral blood compared to 34% GFP+B220+ lymphoid leukemia cells in placebo-treated mice. Furthermore, HHT significantly inhibited in vitro proliferation of K562 and B-lymphoid leukemic cells isolated from mice with B-ALL induced by BCR-ABL wild type and BCR-ABL-T315I resistant to both imatinib and dasatinib. In sum, HHT has an inhibitory activity against CML stem cells, and is highly effective in treating CML and B-ALL induced by BCR-ABL in mice.

The Tumor Suppressor Role of the Msr1 Gene in Cancer Stem Cells of Chronic Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 188-188
Author(s):  
Yaoyu Chen ◽  
Con Sullivan ◽  
Shaoguang Li
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Marrow Cells

Abstract Abstract 188 We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice (Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature Genetics 41:783-792, 2009). We believe that the Alox5 pathway represents a major molecular network in LSCs. Therefore, we decided to further dissect this pathway by comparing gene expression profiles between wild type and Alox5−/− LSCs from CML mice using the DNA microarray analysis. We identified a small group of candidate genes that were changed in expression in the absence of Alox5. Among these genes, we have identified the Msr1 gene and chosen to test the function of this gene in regulating LSC function, because this gene was up-regulated, indicating that it might play a tumor suppressor role in LSCs. In our CML mouse model, we observed that recipients of BCR-ABL transduced Msr1−/− bone marrow cells developed CML much rapidly than recipients of BCR-ABL transduced wide type bone marrow cells. To test whether this accelerated CML is related to abnormal function of LSCs, we carried out a serial transplantation assay by transferring bone marrow cells from primary recipients of BCR-ABL-transduced wild type or Msr1−/− donor bone marrow cells into secondary and next-generation of recipient mice to biologically assess the effect of Msr1 on LSCs. BCR-ABL-expressing wild type leukemia cells from bone marrow of CML mice were only able to transfer CML once, whereas BCR-ABL-expressing Msr1−/− leukemia cells were able to transfer lethal CML for five genrations. This observation indicates that BCR-ABL-expressing Msr1−/− LSCs have markedly increased stem cell function. To further compare the stem cell function, we performed the leukemia stem cell competition assay by 1:1 mixing wild type (CD45.1) and Msr1−/− (CD45.2) bone marrow cells from CML mice. At day 25 or 30 after transplantation, more than 60% and 95% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2+Msr1−/− myeloid leukemia cells, and all these mice developed CML and died of CML derived from Msr1−/− LSCs. To confirm the tumor suppressor role of Msr1 in CML development, we co-expressed BCR-ABL and Msr1 in MSR1−/− bone marrow cells by retroviral transduction, followed by transplantation of these cells into recipient mice. The ectopically-expressed Msr1 in MSR1−/− bone marrow cells rescued the accelerated CML phenotype, and some recipient mice did not even develop the CML. Together, these results demonstrate that Msr1 plays a tumor suppressor role in LSCs. The Msr1 pathway is a novel molecular network in LSCs, and it will be important to fully study this pathway for developing curative therapeutic strategies for CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals KLF4 Controls Leukemic Stem Cell Self-Renewal in MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1231-1231
Author(s):  
Andrew Lewis ◽  
Chun Shik Park ◽  
Monica Puppi ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Epigenetic Mechanisms ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Limiting Dilution

Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1879-1879
Author(s):  
Chun Shik Park ◽  
Ye Shen ◽  
Koramit Suppipat ◽  
Andrew Lewis ◽  
Julie Tomolonis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Parp Cleavage ◽  
Leukemic Stem Cells ◽  
Vitamin K3 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients. Disclosures No relevant conflicts of interest to declare.

BCR/ABL+ CML Stem Cells (CD34+/CD38-) Express High Levels of CD33 and Are Responsive to a CD33-Targeting Drug: a New Potential Concept for Eradication of CML Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3382-3382
Author(s):  
Harald Herrmann ◽  
Sabine Cerny-Reiterer ◽  
Karoline V. Gleixner ◽  
Katharina Blatt ◽  
Susanne Herndlhofer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Chronic Phase ◽  
Normal Bone Marrow ◽  
Leukemic Stem Cells ◽  
Normal Bone ◽  
Advanced Phase

Abstract Abstract 3382 Siglec-3 (CD33) is an established therapeutic target in acute myeloid leukemia (AML). We and others have shown that CD33 is expressed on immature CD34+/CD38- stem cells in AML. We here report that leukemic stem cells obtained from patients with chronic myeloid leukemia (CML) display high levels of CD33, and that CD33 may serve as a potential target in CML. As assessed by multi-color flow cytometry, CD34+/CD38-/CD123+ CML progenitor cells in chronic phase (CP) were found to express significantly higher levels of CD33 compared to normal CD34+/CD38- bone marrow stem cells (figure). By contrast, similar levels of the cell surface antigen MDR1 (CD243) were detected when comparing normal and CML progenitors. In chronic phase (CP) CML, CD33 was found to be expressed homogeneously on most or all CD34+/CD38- stem cells. In patients with accelerated (AP) or myeloid blast phase (BP), CML stem cells also co-expressed CD33, but the levels of CD33 varied from donor to donor, and in one patient, most CML stem cells appeared to be CD33-negative cells. In two patients with CML, CD34+/CD38- cells were highly enriched by cell sorting (purity >98%) and found to contain CD33 mRNA in qPCR analysis. The presence of BCR/ABL and thus the leukemic origin of these cells was confirmed by FISH analysis and PCR. We then examined the effects of the CD33-targeted drug gemtuzumab/ozogamicin (GO) on growth of primary CML cells. As assessed by 3H-thymidine uptake, GO produced growth inhibition in leukemic cells in all patients tested (CP, n=13; AP, n=3). The effects of GO on leukemic cell growth were dose-dependent and occurred at relatively low concentrations, with IC50 values ranging between 1 and 100 ng/ml. GO effects were also seen in precursor-enriched Lin-negative CML cells (n=3). As assessed by Annexin-V staining, GO was found to induce apoptosis in CD34+/CD38- CML progenitor cells. Next we investigated drug combination effects. In these experiments, GO was found to synergize with nilotinib and bosutinib in producing growth inhibition in primary CML cells. In conclusion, CD33 is expressed abundantly on immature CD34+/CD38- progenitor cells in CML. Whether GO can be employed to eradicate residual leukemic stem cells in CML patients alone or in combination with BCR/ABL kinase inhibitors remains at present unknown. Figure: Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Figure:. Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

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