Dual oligopeptides modification mediates arsenic trioxide containing nanoparticles to eliminate primitive chronic myeloid leukemia cells inside bone marrow niches

Abstract The BCR-ABL oncogene plays an essential role in chronic myeloid leukemia (CML). In NOD/scid mice injected with soft agar clones of a human CML cell line (K562), we observed a leukemia syndrome involving not only leukemia but also a severe reduction of normal mouse hematopoiesis (Lin et al., Oncogene, 2001). Some of these mice died of a wasting syndrome that involved suppression of hematopoiesis without extensive tumor cell invasion of the spleen and marrow. In CML patients, since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we postulate that this is an active process mediated by the leukemia cells. The lipocalin 24p3 is secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells (Devireddy et al., Science, 2001). We found that BCR-ABL+ mouse hematopoietic cells induce a persistent secretion of a modified form of 24p3 (21 kDa). Co-culture studies show that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Importantly, BCR-ABL+ hematopoietic cells are resistant to apoptosis under the same conditions. Conditioned medium (CM) from BCR-ABL+ cells expressing anti-sense/siRNA 24p3 or CM mixed with 24p3 antibody have reduced apoptotic activity for target cells. We also found that the expression of the Bcr-Abl oncoprotein and its tyrosine kinase are required for induction of 24p3 expression. Leukemic mice induced by BCR-ABL+ cells expressing anti-sense/siRNA 24p3 have increased levels of normal hematopoiesis (marrow and spleen erythropoiesis and blood platelet levels) and reduced invasion of leukemia cells in marrow and spleen tissues, but the leukemia cells readily invade liver and the abdomen as ascites (Lin et al, Oncogene, 2005). These findings indicate that suppression of normal hematopoiesis in BCR-ABL induced leukemia is an active process involving the apoptotic factor 24p3, raising the possibility that similar factors are involved in BCR-ABL+ CML patients. We have found that the K562 clones (Lin et al. 2001) have enhanced expression of NGAL (neutrophil gelatinase-associated lipocalin, human homologue of 24p3) transcripts compared to uncloned K562 cells. We generated additional soft agar K562 clones, each with different expression levels of NGAL transcripts. NOD/scid mice injected with the clone (C5) of K562 cell line expressing a high level of NGAL had severe depression of hematopoiesis and significantly shorter survival time as compared with mice injected with parental K562 cells and a clone (C6) expressing a low level of NGAL. Co-culture studies showed that the C5 K562 clone also induced apoptosis in BCR-ABL negative cells. We detected two glycosylated forms of NGAL/24p3 migrating at 24 kDa and 21 kDa on SDS-PAGE. The 21 kDa form is the major form in CM from mouse BCR-ABL+ cells and K562 clones. Our preliminary data with CML patient samples showed that levels of 21 kDa NGAL protein in bone marrow fluid correlated with BCR-ABL/ABL ratio. Further studies with more patient samples are ongoing to confirm the role of NGAL in suppressing normal hematopoiesis in CML patients and to determine the structural change(s) that leads to the modified form of 24p3/NGAL secreted by CML cells.

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

Blood ◽

10.1182/blood-2019-129768 ◽

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.

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

Blood ◽

10.1182/blood.v114.22.188.188 ◽

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.

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Bone marrow-derived mesenchymal stromal cells expressing interferon-gamma inhibit proli-feration of chronic myeloid leukemia cells in vitro

Cytotherapy ◽

10.1016/j.jcyt.2013.01.212 ◽

2013 ◽

Vol 15 (4) ◽

pp. S54-S55

Author(s):

L.C. Liew ◽

A. Maha ◽

P.P. Chong ◽

C.F. Leong ◽

S. Fadilah ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Interferon Gamma ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Leukemia Cells

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Faculty Opinions recommendation of Kindlin-3 loss curbs chronic myeloid leukemia in mice by mobilizing leukemic stem cells from protective bone marrow niches.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738672390.793583306 ◽

2021 ◽

Author(s):

Daniela Krause

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Stem Cells ◽

Bone Marrow Niches

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Effective Targeting Of Chronic Myeloid Leukemia Initiating Activity With The Combination Of Arsenic Trioxide and Interferon Alpha

Blood ◽

10.1182/blood.v122.21.5171.5171 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5171-5171

Author(s):

Rabab El Eit ◽

Iskandarani Ahmad ◽

Jessica Saliba ◽

Mark Jabbour ◽

Rami Mahfouz ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Arsenic Trioxide ◽

Interferon Alpha ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Clinical Activity ◽

Retroviral Transduction

Abstract Imatinib is the standard of care in the treatment of Chronic Myeloid Leukemia (CML) patients. However, imatinib is not curative since most patients who discontinue therapy will relapse, stressing the need for novel therapies that target leukemia initiating cells (LIC). Arsenic trioxide induces complete remission in patients with acute promyelocytic leukemia (APL). We previously demonstrated that, through full degradation of PML-RARA, the combination of arsenic and retinoic acid eradicates APL LICs and cures the disease. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Moreover, in a murine transplantation model of CML, we show that arsenic/IFN prolonged survival of primary leukemic mice and sharply diminished transplantation of CML cells in secondary recipients, pointing to exhaustion of CML LICs. Our results also suggest that IFN/arsenic may clear LICs independently from oncoprotein catabolism. Interestingly, arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib.These studies plea for a clinical exploration of this combination, knowing that IFN and arsenic have both shown clinical activity in CML, alone or in combination with imatinib. Disclosures: No relevant conflicts of interest to declare.

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