Letters to the Editor 2559 2 Mollace V, Nottet HS, Clayette P, Turco MC, Muscoli C, Salvemini 5 Weiss RA. The discovery of endogenous retroviruses. Retrovirology D et al. Oxidative stress and neuroAIDS: triggers, modulators and 2007; 3: 67. novel antioxidants. Trends Neurosci 2001; 24: 411–416. 6 Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. 3 Tristem M. Identification and characterization of novel human A rapid and simple method for measuring thymocyte apoptosis by endogenous retrovirus families by phylogenetic screening of the human propidium iodide staining and flow cytometry. J Immunol Methods genome mapping project database. JVirol2000; 74: 3715–3730. 1991; 139: 271–279. 4 Nelson PN, Hooley P, Roden D, Davari Ejtehadi H, Rylance P, 7 Bonelli P, Petrella A, Rosati A, Romano MF, Lerose R, Pagliuca MG Warren P et al. Human endogenous retroviruses: transposable et al. BAG3 protein regulates stress-induced apoptosis in normal elements with potential? Clin Exp Immunol 2004; 138: 1–9. and neoplastic leukocytes. Leukemia 2004; 18: 358–360.

Pleiotropic p27Kip1, BCR-ABL and leukemic stem cell: the trio in concert

Leukemia (2007) 21, 2559–2561; doi:10.1038/sj.leu.2404842; rendering them as putative therapeutic targets. Nevertheless, published online 5 July 2007 prioritization of such extra BCR-ABL secondary transformation- derived therapeutic targets is not only crucial for preventive medicine but to a better understanding of the molecular The mammalian cyclin-dependent kinase inhibitor (CDKI) p27 is mechanisms of CML blast transformation. Interestingly, several a potent regulator of cell cycle arresting transition from G1 to S lines of evidence indicate involvement of deregulated p27 as phase. In contrast to earlier opinion of mere cytoplasmic one of such crucial events in leukemogenesis (Table 1). incapacitation of p27, there is a growing body of evidence Emerging evidence indicates that BCR-ABL-mediated CML suggesting that p27 could be transformed from a native CDKI CD34 þ cell proliferation is intertwined with the nucleocyto- into a dynamic oncoprotein at the expense of its extranuclear plasmic distribution of p27. BCR-ABL-induced constitutive localization in leukemic cells. Emerging concepts also indicate activation of PI3K in CML cells causes p27 T157 phosphoryla- that there are certain resident leukemic stem cells (LSC) in bone tion and results in its cytoplasmic sequestration. PI3K can also marrow milieu, which possess exuberant self-renewal charac- promote downregulation of p27 both by suppression of synthesis teristics and they eventually lead to conventional therapeutic and stimulation of protein degradation. Indeed, it has been þ þ recalcitrance. Given that BCR-ABL-containing CD34 cells demonstrated that unlike in normal CD34 cells, b1-integrin þ successfully persist in chronic myeloid leukemia (CML) patients engagement on CML CD34 cells does not inhibit G1–S with cytogenetic remission under imatinib, it is necessary to elucidate additional incongruous molecular program that might have significant therapeutic implications. In this connection, considering the functional nexus among p27, BCR-ABL and a Table 1 Summary of studies indicating deregulation of p27 in transmembrane glycoprotein CD44 in the regulation of homing, leukemogenesis engraftment and proliferation of LSCs together with the recent findings that p27 can promote cellular migration and metastasis Role of p27 Proposed mechanism Reference in several other malignancies,1–4 it could be pertinent to address the possibilities of p27 pleiotropism in regenerative and CD44 ligation CD44 ligation-dependent Williams and and cell cycle inhibition of AML cell Cancelas,1 preventive medicine. arrest proliferation is accompanied Gadhoum et al.2 During G0- to S-phase transition, p27 proteolysis requires by enhanced p27 stability intact p27-cyclin-cdk trimeric motif. Cdk2-dependent phosphor- Abnormal CML BCR-ABL induces elevated Jiang et al.7 ylation of p27 at T187 promotes Skp1-Cul1-F-box (SCFSkp2) proliferation levels of p27 sequestered into the cytoplasm, leading to ubiquitin Ligase-mediated degradation of p27. In early G1 T187- independent p27, proteolysis can also take place that involves loss of b1-integrin-mediated CML proliferation inhibition Skp2-dependent and Skp2-independent mechanisms. However, Phosphorylation Cdk inhibitory activity and Grimmler et al.8 proteolysis of cytoplasmic pool of p27 in G1 sometimes involves and inactivation stability of p27 are negatively a different Kip1 ubiquitination promoting complex ubiquitin affected by Lyn kinase- ligase. Furthermore, in early G1, phosphatidylinositol-3-kinase dependent Y88 phosphorylation (PI3K)-dependent phosphorylation at T157 on the nuclear 7 localization signal of p27 promotes cytoplasmic sequestration. Phosphorylation, BCR-ABL activates PKB/Akt, Jiang et al., cytoplasmic the later phosphorylates p27 Grimmler et al.,8 In addition, molecular control of transcription, mRNA stabiliza- sequestration and at T157 and therefore impairs Cheng9 tion and protein degradation might all act in concert to impinge ubiquitination its nuclear import and G1 on the biological functions of p27. arrest. BCR-ABL also CML is a clonal, myeloproliferative and multiphasic stem cell induces expression of Skp2 disorder of hematopoietic development. Transformation by and the later promotes p210BCRÀABL is a complex mechanism, implicating enhanced ubiquitination-dependent p27 degradation proliferation and inhibition of apoptosis, but conserved differ- Acute Acute overexpression of wild Sengupta et al.11 entiation particularly in the initial chronic phase of the overexpression type and T187A, T257A p27 disease.5,6 Paradoxically, blast crisis CML is marked with acute and cell cycle in BCR-ABL+ CML cells differentiation blockages. Although BCR-ABL has been one of arrest induces cell cycle arrest and the primary transforming events responsible for CML develop- proliferation inhibition ment, there are other associated deregulated molecular check- Abbreviations: AML, acute myeloid leukemia; CML, chronic myeloid points that play important role in the blast transformation, thus leukemia.

Leukemia Letters to the Editor 2560 progression and the proliferative advantage is synchronized with Another important aspect in the context of LSC is how p27 cytoplasmic sequestration of p27.7 Therefore, since BCR-ABL is can control their homing, engraftment and niche-dependent considered as a hallmark of CML development, the manner in maintenance of the tumor burden. Recent studies demonstrate which it regulates p27 stability warrants future investigations. that BCR-ABL-expressing LSCs depend to a larger extent on Likewise, whether BCR-ABL is responsible for relocation and CD44 for their homing and engraftment into bone marrow functional inactivation or constitutive activation of other microenvironmental niche than do normal HSCs; therefore, molecules that might regulate cell growth needs to be CD44 blockage might be beneficial in autologous transplanta- examined. tion for CML.1 Notably, CML cells do have functional defects in Furthermore, p27 can also be deregulated by constitutive Y88 b1-integrin and stromal cell-derived factor-1-mediated adhesion phosphorylation in BCR-ABL-expressing leukemias. Recently it and transmigration, which are indicative of alternative adhesive has been shown that nonreceptor cytoplasmic tyrosine kinase mechanisms necessary for LSC engraftment. Indeed, BCR-ABL Lyn induces phosphorylation of p27 (p27-cyclin-cdk trimeric induces expression of functional E-selectin ligands on LSCs motif) in vitro in its N-terminus cdk-inhibitory domain at Y88.8 partially by upregulation of the hyaluronan receptor CD44. Eventually p27 is transformed into a cdk-substrate and thus is CD44 ligation can also lead to eradication of acute myeloid triggered for SCFSkp2-dependent degradation. Although the leukemia stem cells that includes pro-differentiation.1 Then intrinsic flexibility of p27 has been ascribed for this phosphory- again, CD44-dependent differentiation therapy is accompanied lation, it is still to validate whether such mechanism really exists by enhanced p27 stability.2 Therefore considering deregulated in vivo. Given that blast crisis CML is accompanied by p27 associated with CML, it might be important to investigate enhanced Lyn expression, future studies could be considered the BCR-ABL-p27-CD44 axis in CML stem cell–niche interac- to address whether other members of the Src family of tyrosine tion (Figure 1). kinases, such as Fyn or Yes, can exert similar effect. In addition, In addition, p27 has been suggested to regulate cellular even in the blast-phase CML cells, there could be a moderate migration in a spatiotemporal manner, which is an important pool of p27 sequestered in the cytoplasm and it also remains to aspect in tumor metastasis. Interestingly, this notion gets a be seen whether that could be putative targets of other sudden impetus when it was reported that cytoplasmic p27 nonreceptor kinases. could interfere with RhoA activation by guanine-nucleotide- Several studies carried out on malignant hematopoiesis over exchange factors through its C terminus and thereby regulating the last few years have bolstered putative involvement of cellular migration.4 Rho proteins regulate and coordinate the dynamic LSC compartments in chronic and acute leukemias. cytoskeletal remodeling that underlies changes in cell adhesion Considering BCR-ABL-induced cytoplasmic mislocalization of and migration. CML is linked with modifications of the actin p27 in CML cells one can hypothesize whether this is a mere cytoskeleton, and BCR-ABL is preferentially associated with F- aftermath of CDKI inactivation or this sequestration is a actin and its distribution changes in migrating cells. Recently it paradigm in CML stem cell biology. In other words, is deregulated redistribution of p27 in CML simply analogous to the loss of CDKI and/or tumor suppressive function or oncoprotein-like functions that are programmatically acquired in CML cytoplasm? Similarly, considering the studies based on the analysis of normal/tumor stem cell samples and CDKI knockout mouse models, one can not rule out the possibilities of deregulation of other CDKIs in LSC transformation.9 It has been suggested that downregulation of p18 along with disruption of p15 and p16 through promoter hypermethylation could act as secondary transforming events that might set the platform of LSC transformation to outcompete normal hematopoiesis. Further- more, given that both normal and tumor stem cells can share Bmi-1- and b-catenin-mediated self-renewal and p16/p19 can partially mediate the role of Bmi-1 in the cell cycle of stem cells, it remains to be elucidated whether b-catenin or other self- renewal pathways might involve CDKI functioning during CML development.9 Recently we have demonstrated that deregula- tion and cross-talk among Sonic hedgehog, Wnt, Hox and Notch signaling during CML progression accompanies with dynamic changes in p21 and p27.10 Moreover, acute over- expression of a stable p27 molecule had a promising edge in CML therapeutics.11 It is thus becoming increasingly evident that p27 and other CDKIs can play distinct roles, depending on Figure 1 Model to show integration of different BCR-ABL-p27 the sub-compartments of the hematopoietic cascade they axes in CML stem cell regulation. BCR-ABL induces expression of CD44 in CML stem cells. The transmembrane glycoprotein CD44 can belong. In addition, epigenetic control of gene expression and stabilize p27 in an intriguing manner (dotted line). In addition, nuclear microRNA-mediated post-transcriptional regulation at the pri- localization of p27 is usually inhibited by BCR-ABL resulting mitive stem cell level might produce an additional degree of cytoplasmic pool of p27. Taken together, BCR-ABL-p27-CD44 axis complexity in making regulated/deregulated self-renewal and might be responsible for abnormal CML stem cell proliferation. On the differentiation associated with normal and malignant hemato- other hand, BCR-ABL can lead to cytoplasmic remodeling and CML poiesis.12 Nonetheless, LSC might exploit these subtleties stem cell homing by RhoGTPase activation. Furthermore, p27 can regulate cell migration and metastatic potential of other tumors necessary for hematopoietic stem cell (HSC) maintenance in through RhoA interaction (dotted line). All together, BCR-ABL-p27- making deregulated p27, be it as a secondary transforming event RhoGTPase axis can regulate CML stem cell homing, engraftment and but necessary for CML development. proliferation. CML, chronic myeloid leukemia.

Leukemia Letters to the Editor 2561 has been reported that, in contrast to p190BCRÀABL that is regret for not citing several original references owing to space normally found in Ph þ ALL, CML associated p210BCRÀABL by constraints. virtue of its unique Dbl/Plecstrin-homology domain, can bind to small GTPases of the Rho family both in vitro and in vivo.3 This A Sengupta and S Banerjee in turn gives rise to Rho activation, which might be responsible Structural Genomics Section and Biophysics Division, Saha Institute of Nuclear Physics, Kolkata, India for promoting cellular motility. Taken together, it need to be E-mail: [email protected] substantiated whether or not these events do indeed contribute to malignant transformation; however, considering BCR-ABL- induced abundant pool of p27 in the CML cytoplasm, one can at References least speculate the involvement of BCR-ABL-p27-RhoGTPase axis in CML blast transformation (Figure 1). 1 Williams DA, Cancelas JA. Leukemia: niche retreats for stem cells. Nature 2006; 444: 827–828. Past few decades have witnessed considerable advancement 2 Gadhoum Z, Leibovitch MP, Qi J, Dumenil D, Durand L, in identifying putative phosphorylation hotspots in p27 that are Leibovitch S et al. CD44: a new means to inhibit acute myeloid important for cell cycle regulation. The N-terminus portion of leukemia cell proliferation via p27Kip1. Blood 2004; 103: p27 is recruited to cyclin–cdk complexes and results in cell 1059–1068. cycle arrest and it is the C-terminus portion that can promote 3 Harnois T, Constantin B, Rioux A, Grenioux E, Kitzis A, cellular motility and thus metastatic potential. Intriguingly, these Bourmeyster N. Differential interaction and activation of Rho family GTPase by p210bcrÀabl and p190bcrÀabl. Oncogene 2003; two functions are virtually partitioned by a single parameter: the 22: 6445–6454. address of p27 at a given point of time. Future studies could be 4 Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM. p27Kip1 directed to unravel the individual domain contribution by using modulates cell migration through the regulation of RhoA activa- respective knockout animal models, and it would be important tion. Genes Dev 2004; 18: 862–876. to look upon whether tampering p27 in such a manner can have 5 Mughal T, Cortes J, Cross NC, Donato N, Hantschel O, Jabbour E any consequences on cancer predisposition. Furthermore, et al. Chronic myeloid leukemia – some topical issues. Leukemia 2007; E-pub May 10. different CDKIs control diverse array of functions for HSC 6 Diaz-Blanco E, Bruns I, Neumann F, Fischer JC, Graef T, Rosskopf M maintenance. However, we know very little about how these et al. Molecular signature of CD34(+) hematopoietic stem and different CDKIs differentially regulate quiescence, self-renewal progenitor cells of patients with CML in chronic phase. Leukemia and homing, three important aspects of regenerative medicine, 2007; 21: 494–504. 7 Jiang Y, Zhao RCH, Verfaillie CM. Abnormal integrin-mediated of normal and malignant HSCs. To conclude, we are only + beginning to appreciate the underlying importance of p27 and regulation of chronic myelogenous leukemia CD34 cell prolifera- tion: BCR/ABL upregulates the cyclin-dependent kinase inhibitor, other cell cycle inhibitors in the context of LSCs. Indeed, how p27Kip1, which is relocated to the cell cytoplasm and incapable such an intrinsically unstructured p27 protein governs CML of regulating cdk2 activity. Proc Natl Acad Sci USA 2000; 97: biology need to be explored further. 10538–10543. 8 Grimmler M, Wang Y, Mund T, Cilensek Z, Keidel EM, Waddell MB et al. Cdk-inhibitory activity and stability of p27Kip1 are directly Acknowledgements regulated by oncogenic tyrosine kinases. Cell 2007; 128: 269–280. 9 Cheng T. Cell cycle inhibitors in normal and tumor stem cells. Oncogene 2004; 23: 7256–7266. We thank Drs Tony Hunter and Joyce Slingerland for providing 10 Sengupta A, Banerjee D, Chandra S, Banerji SK, Ghosh R, Roy R p27 constructs used in our studies. Grateful acknowledgement is et al. Deregulation and cross talk among Sonic hedgehog, Wnt, earnestly due to Drs David Scadden, Jon Aster, Tom Kadesch, Hox and Notch signaling in chronic myeloid leukemia progres- Hans Clevers, Chris Albanese, Toshiyuki Sakai, Jacqueline sion. Leukemia 2007; 21: 949–955. Bromberg and John Clifford for various molecular constructs that 11 Sengupta A, Banerjee D, Chandra S, Banerjee S. Gene therapy for BCR-ABL+ human CML with dual phosphorylation resistant p27kip1 were used to decipher crosstalk among self-renewal-associated þ and stable RNA interference using an EBV vector. J Gene Med signaling pathways in CML CD34 cells. This study would not 2006; 8: 1251–1261. have been possible without their generous cooperation. We also 12 Oakley EJ, Van Zant G. Unraveling the complex regulation of stem thank Saha Institute of Nuclear Physics for financial assistance and cells: implications for aging and cancer. Leukemia 2007; 21: 612–621.

No evidence for amplification of V617F JAK2 in myeloproliferative disorders

Leukemia (2007) 21, 2561–2563; doi:10.1038/sj.leu.2404845; up study, the same group found that this phenomenon was most published online 5 July 2007 prevalent in PV, with more than one-third of mutation-positive cases apparently having more than two V617F copies/cell.3 Increased V617F copy number has been described in cell lines,4 Acquired uniparental disomy following mitotic recombination but has not thus far been verified in patients. results in homozygosity for the V617F JAK2 mutation in a To determine if elevated JAK2 copy number could be detected proportion of patients with myeloproliferative disorders (MPD), with an alternative methodology, we developed a test based on particularly polycythemia vera (PV) and myelofibrosis (MF).1 multiplex ligation-dependent probe amplification (MLPA), an Recently, Hammond et al.2 in a letter in Leukemia indicated that established technique for detecting DNA copy number a proportion of MPDs harbor more than two copies of V617F changes.5–7 Four MLPA probe pairs targeting JAK2 exons 3, 9, JAK2 and suggested that disease evolution may involve further 16 and 20 were designed, so that they could be combined with gene duplication associated with genetic instability. In a follow- the commercially available telomere MLPA probe mix (P036B

Leukemia