Autonomous multi-lineage differentiation in vitro of primitive CD34+ cells from patients with chronic myeloid leukemia

Abstract Primitive quiescent CD34+ cells in chronic myeloid leukemia (CML) are relatively resistant to the tyrosine kinase inhibitors imatinib and dasatinib, which may explain the persistence of detectable BCR-ABL transcripts following treatment with these agents. Conversely, allogeneic stem cell transplantation (SCT) can eradicate residual CML, suggesting that quiescent stem cells are eliminated by graft-versus-leukemia (GVL) effects. We studied the progeny of CD34+ cells after 4 days culture in serum-free media supplemented with interleukin-3, interleukin-6, stem cell factor, granulocyte-colony stimulating factor and Flt-3 ligand in 14 CML patients (8 chronic phase, 6 advanced phase) who subsequently received T cell depleted SCT from their HLA-identical sibling donors. Cycling CD34-negative and CD34+, and non-cycling quiescent CD34+ CML cells were isolated by fluorescence activated cell sorting. Fluorescence in situ hybridization in 4 representative CML patients revealed over 80% BCR-ABL positivity in both quiescent and cycling CD34+ and CD34-negative populations. Using real-time quantitative polymerase chain reaction, we found the expression of BCR-ABL, and leukemia-associated antigens (LAA), WT1, PR3 and ELA2, were the same in both cycling and quiescent CD34+ cell populations in CML. LAA expression was not significantly different when compared with similarly cultured CD34+ cells from healthy donors. Pre-SCT quiescent CD34+ cells from CML patients were lysed by natural killer (NK) cells from their donors but were less susceptible than their cycling CD34+ and CD34-negative counterparts. Purified donor NK cells (n=7) expanded after 11–13 days culture with interleukin-2 and irradiated EBV-LCL lysed quiescent CD34+ CML cells as well as their cycling CD34+ and CD34-negative progeny. Previous studies have demonstrated that bortezomib can sensitize malignant cells to NK-cell tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Lundqvist et al Cancer Res. 2006 Jul 15;66(14):7317–25). Addition of bortezomib 10nM to CD34+ cell cultures enhanced cytotoxic effects of expanded donor NK cells on quiescent CD34+ CML cells. As observed with other malignancies, this enhanced sensitivity to NK-cytotoxicity correlated with increased expression of TRAIL receptors DR4 and DR5 on the surface of CD34+ quiescent cells, compared with cycling CD34+ or CD34-negative cells. Bortezomib treatment did not significantly affect the expression of MHC Class I, MIC A/B or Fas (CD95) on CD34+ quiescent or cycling cells. These results suggest that adoptive transfer of in vitro expanded donor NK cells with concomitant administration of bortezomib to the recipient may enhance cytotoxicity to quiescent CD34+ cells and may improve NK-mediated GVL effects. This may be particularly applicable to CML patients who are increasingly transplanted in more advanced stage disease, and so are at a greater risk of relapse post-SCT.

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Statins Enhance the Molecular Response in Chronic Myeloid Leukemia When Combined with Tyrosine Kinase Inhibitors

Cancers ◽

10.3390/cancers13215543 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5543

Author(s):

Hyeok-Jae Jang ◽

Young-Min Woo ◽

Kazuhito Naka ◽

Jong-Ho Park ◽

Ho-Jae Han ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Molecular Response ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Cd34 Cells

Previous studies have suggested that statins can be repurposed for cancer treatment. However, the therapeutic efficacy of statins in chronic myeloid leukemia (CML) has not yet been demonstrated. In this study, we retrospectively evaluated the outcomes of 408 CML patients who underwent imatinib therapy. The deep molecular response rates in patients treated with the statin/TKI combination were significantly higher than those in patients treated with TKI alone (p = 0.0016). The statin/TKI combination exerted potent cytotoxic effects against wild-type and ABL1 mutant CML, BaF3, and K562/T315I mutant cells. Furthermore, the statin/TKI combination additively inhibited the colony-forming capacity of murine CML-KLS+ cells in vitro. In addition, we examined the additive growth-inhibitory effects of the statin/tyrosine kinase inhibitor (TKI) combination against CML patient-derived CD34+ cells. The growth-inhibitory effects of the statin/imatinib combination against CD34+/CML primary cells were higher than those against CD34+/Norm cells (p = 0.005), suggesting that the combination of rosuvastatin and imatinib exerted growth-inhibitory effects against CML CD34+ cells, but not against normal CD34+ cells. Furthermore, results from RNA sequencing of control and statin-treated cells suggested that statins inhibited c-Myc-mediated and hematopoietic cell differentiation pathways. Thus, statins can be potentially repurposed to improve treatment outcomes in CML patients when combined with TKI therapy.

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Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib

Blood ◽

10.1182/blood-2008-05-158253 ◽

2009 ◽

Vol 113 (4) ◽

pp. 875-882 ◽

Cited By ~ 44

Author(s):

Agnes S. M. Yong ◽

Keyvan Keyvanfar ◽

Nancy Hensel ◽

Rhoda Eniafe ◽

Bipin N. Savani ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Myeloid Leukemia ◽

Nk Cell ◽

Receptor Expression ◽

Pharmacologic Therapy ◽

Trail Receptors ◽

Cd34 Cells

AbstractPrimitive quiescent CD34+ chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34+ cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34+ populations. Quiescent CD34+ cells from CML patients were less susceptible than their cycling CD34+ and CD34− counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34+ CML cells had higher surface expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34+ CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell–mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

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Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3

Blood ◽

10.1182/blood.v97.3.720 ◽

2001 ◽

Vol 97 (3) ◽

pp. 720-728 ◽

Cited By ~ 111

Author(s):

Tessa L. Holyoake ◽

Xiaoyan Jiang ◽

Heather G. Jorgensen ◽

Susan Graham ◽

Michael J. Alcorn ◽

...

Keyword(s):

Growth Factors ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Philadelphia Chromosome ◽

Leukemic Cells ◽

Regulation Of Expression ◽

Ki 67 ◽

Interleukin 3 ◽

Cd34 Cells

Abstract It was previously shown that patients with chronic myeloid leukemia (CML) have a rare but consistently detectable population of quiescent (G0) leukemic (Philadelphia chromosome–positive and BCR-ABL–positive [BCR-ABL+]) CD34+ cells. In the study described here, most such cells expressed a primitive phenotype (CD38−, CD45RA−, CD71−, and HLA-DRlo) and cultures of these cells containing growth factors produced ultimately larger, but initially more slowly growing clones than do cultures of initially cycling CD34+ leukemic cells. Initially quiescent leukemic cells expressing BCR-ABLproliferated in single-cell cultures in the absence of added growth factors, thereby demonstrating their ability to spontaneously exit G0 and enter a continuously cycling state. Interestingly, on isolation, few of these quiescentBCR-ABL+ cells contained either interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF) transcripts, whereas both were present in most cyclingBCR-ABL+ CD34+ cells. However, after 4 days of culture in the absence of added growth factors and in association with their entry into the cell cycle (as indicated by up-regulation of Ki-67 and cdc25 transcripts), IL-3 transcripts became detectable. These findings show that entry of leukemic (BCR-ABL–expressing) progenitors into a quiescent (G0) state in vivo is highest among the most primitive leukemic cell populations, associated with a down-regulation of IL-3 and G-CSF gene expression, and spontaneously reversible in association with up-regulation of IL-3 expression. These results highlight the potential physiologic relevance of quiescent CML progenitors, even in treated patients, in whom these cells would be predicted to have a proliferative advantage over their quiescent normal counterparts when cytokine concentrations are low.

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