In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34 ؉ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin ؊ CD34 ؉ ) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable highresolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ؎ STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34 ؉ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy. IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the t(9;22) chromosome translocation that, in turn, creates the BCR-ABL oncogene. [1][2][3] The fusion gene product is a p210 oncoprotein containing a constitutively active tyrosine kinase that confers certain growth advantages to the Philadelphia-positive (Ph ϩ ) clone compared with normal hematopoietic cells. 4 We have demonstrated recently the existence of a population of rare, primitive, quiescent stem cells in all chronic-phase CML patient samples, whether derived from peripheral blood or bone marrow. These stem cells are predominantly Ph ϩ , express high levels of CD34 ϩ but lack the markers CD38, CD45RA, or CD71, and can spontaneously exit G 0 to enter a continuously proliferating state, either in vitro or to produce Ph ϩ progeny in immunocompromised mice in vivo. 5,6 Many cancers are treated with relatively nonselective cytotoxic drugs that affect normal and malignant cells. Because most available chemotherapeutic agents show some degree of S-phase specificity, cells that are not actively dividing may prove resistant to such drugs. This raises the possibility that the quiescent leukemic cells we have identified in patients with CML are likely to survive standard chemotherapy r...

IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder originating in a hematopoietic stem cell. It is characterized by the Philadelphia (Ph) chromosome, 1 arising from a reciprocal translocation between the long arms of chromosomes 9 and 22, resulting in fusion of the BCR gene on chromosome 22 with the ABL oncogene on chromosome 9, with expression of its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. 2 The deregulated BCR-ABL tyrosine kinase activity is essential for its transforming ability, 3 resulting in phosphorylation of cellular substrates and activation of signal transduction pathways, including the RAS-ERK cascade, JAK-STAT, PI3-kinase, c-Myc, c-CBL, and CrKL, affecting cell growth, stromal interaction, and apoptosis. [4][5][6] Imatinib mesylate (IM; Novartis Pharma, Basel, Switzerland) is a tyrosine kinase inhibitor (TKI) active against ABL, c-KIT, and PDGFR, acting by competitive inhibition of adenosine triphosphate binding to the tyrosine kinase. 7 Despite an impressive rate of durable complete cytogenetic response in chronic phase CML patients treated with IM, 8 only a minority of patients achieve complete molecular remission. 9 At least 2 mechanisms of resistance appear to account for residual disease in these patients: the innate insensitivity of primitive quiescent CML stem/progenitor cells, which greatly overexpress BCR-ABL, to IM 10,11 and BCR-ABL kinase mutations, 12 which are present before IM therapy in a subgroup of patients and result in IM resistance. 13,14 However, there is currently no direct evidence that these resistance mechanisms, observed in vitro, are responsible for residual disease in IM-treated patients.Two main strategies to overcome IM resistance have emerged. These are the development of second generation TKIs 15,16 and the use of IM in drug combinations. 17 Dasatinib (Sprycel, formerly BMS-354825; Bristol-Myers Squibb, Stamford, CT) is an oral, multitargeted inhibitor of BCR-ABL and SRC kinases, with greatly improved potency against wild-type (WT) BCR-ABL, capable of binding all known BCR-ABL kinase mutants resistant to IM except T315I. 15 In our previous work, although dasatinib induced durable inhibition of BCR-ABL in primitive progenitor cells from CML patients compared with either IM or nilotinib, which were ineffective, 11,18 none of these TKIs targeted the most primitive, quiescent CML stem/progenitor cells. Of a wide range of rational drug combinations, 17 the only agent to synergize with IM against these cells was the cytostatic farnesyltransferase inhibitor (FTI) lonafarnib (Schering-Plough). 17 FTIs inhibit oncogenic RAS and have entered clinical trials in solid tumors and acute leukemias. [19][20][21] However, the observed antitumor effects of FTIs are not solely the result of RAS inhibition 22,23 ; they may also act by inhibiting farnesylation of other proteins. 24,25 BMS-214662 is a cytotoxic FTI 26 that produces potent tumor regression and curative responses in human tumor xenografts and transgenic tumor mode...

Summary:suppression. Finally, culture conditions may be developed which could be applied to gene transduction protocols. We have previously demonstrated that CD34 + cells, Most investigators initiate stem/progenitor cell expansion selected from peripheral blood progenitor cells (PBPC), with CD34 + cells purified by positive selection since these can be expanded in ex vivo culture and can be infused cells appear to be superior to unselected material. In in tandem with unmanipulated PBPC with little or no addition, up to a 4.0 log depletion of contaminating tumour toxicity. In this study, four patients (two non-Hodgkin's cells can be obtained using CD34 selected starting populymphoma (NHL), two multiple myeloma (MM)) lations. 7 A recent study suggested that any remaining received myeloablative conditioning prior to stem cell tumour cells do not increase in number during ex vivo rescue using ex vivo expanded cells alone. The two expansion, and this may represent a further purging patients with NHL received cyclophosphamide and total effect. 8,9 The first clinical studies using progenitor cells body irradiation (CY/TBI) and the two patients with expanded ex vivo have now been reported. 5,6,10,11 In only MM, busulphan and melphalan (Bu/M). One case one of these studies, in a small number of patients, stem received an inadequate CFU-GM dose, despite expancell rescue was performed using only the expanded cells, sion, and in one case the expanded cells were contamiwithout unmanipulated 'back-up'. 5 Brugger et al 5 expanded nated. No definitive conclusions may therefore be drawn one tenth of a standard peripheral blood progenitor cell concerning engraftment in these two cases. However, (PBPC) product prior to infusion. Six patients were transthe other two cases received high doses of committed planted with expanded cells only, and five of six experiprogenitors. Following infusion of the expanded enced rapid engraftment following high-dose, but nonmaterial, all four patients failed to show sustained myeloablative, chemotherapy. The remaining patient died neutrophil engraftment and none showed evidence of prior to the anticipated time of engraftment. platelet engraftment. Back-up, unmanipulated PBPC In our previous study, we established the safety and were therefore infused on days 14, 34, 32 and 28 and feasibility of transfusing CD34 + cells which had been selecsubsequently all four cases achieved satisfactory ted from cryopreserved PBPC and then expanded ex vivo. 6 engraftment of both neutrophils and platelets. In conTen patients received у20 × 10 4 CFU-GM/kg unmanipclusion, we feel that, CD34 + cells, expanded ex vivo using ulated PBPC in addition to an aliquot of expanded cells the conditions described in this report, may not provide (range 33-279 × 10 4 CFU-GM/kg). There were no toxic durable engraftment following fully myeloablative effects from the progenitor cells which had been generated conditioning.ex vivo. 6 Keywords: ex vivo expansion; CD34 positive; PBPC;The primary objective of this second study was to det...

Recent studies indicate that a rare population of primitive quiescent BCR-ABL þ cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3 0 kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34 þ leukemic cell population from chronic phase CML patients. However, for quiescent CD34 þ leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.