Page 1240 - Hematology_ Basic Principles and Practice ( PDFDrive )

1086 Part VII Hematologic Malignancies

and platelets. Populations of cells enriched for HSCs, common on the cell surface of platelets and Mks in MPNs has been reported
myeloid progenitors (CMPs), granulocyte–macrophage progenitors to be due to both a reduction in the recycling and maturation of
(GMPs), and megakaryocytic–erythroid progenitors (MEPs) from the receptor as well as an increase in MPL proteasomal degradation
patients with PV have been analyzed for the presence of the mediated by JAK2V617F protein. Furthermore, JAK2V617F and
JAK2V617F mutation. JAK2V617F was detected in HSCs, CMPs, activated STAT5 have been shown to increase the expression of
GMPs, and MEPs from patients with PV, supporting that PV is a 6 phosphofructokinase/fructose-2,6 bisphosphatase 3 (PFKFB3),
disorder that arises in HSCs and involves the myeloid, erythroid, and which controls glycolytic flux through 6-6-phosphofructo-1-kinase.
megakaryocytic lineages. These data indicate that JAK2V617F in PV PFKB3 is required for JAK2V617F-dependent lactate production,
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originates in lymphomyeloid progenitor cells. Analyses of hemato- oxidative metabolic activity, and glucose activity, thereby promoting
poietic cells from PV patients has suggested that JAK2V617F ampli- cell proliferation. It is important to note that many other oncogenic
fies the terminal stages of hematopoiesis but not the more primitive tyrosine kinases activate the same signal transduction pathways, and
hematopoietic stem/progenitor cells. In mouse models, however, the role and requirement for each of these signaling pathways in
JAK2V617F has been shown to lead to the amplification of both stem the transformation of hematopoietic cells by JAK2V617F remain
and progenitor cells, providing them with a competitive advantage unknown. Although once thought to reside strictly in the cytoplasm
compared with their normal counterpart through increased cell of cells, a growing body of evidence indicates that both JAK1 and
cycling and a reduced rate of apoptosis resulting in the emergence of JAK2 are present in the nucleus of certain cells under conditions asso-
the MPN phenotype. JAK2V617F may give only a subtle advantage, ciated with high rates of cell proliferation. Nuclear JAKs have been
which will require several years for mutated stem and progenitor cells reported to affect gene expression by activating other transcription
to predominate in humans over their normal counterparts. In man, factors besides STATs and influencing epigenetic events by phos-
other mutations such as ASXL1, DNMT3a, and TET2 may further phorylating H3 and activating global gene expression. JAK2V617F
alter the biology of the JAK2V617F-mutated clone in a manner that may alter chromatin structure by selectively phosphorylating the
affects phenotype as well as disease evolution. arginine methyltransferase PRMT5, impairing PRMT5 methyltrans-
ferase activity by negatively affecting its association with methylsome
Structural and Functional Aspects of JAK2V617F- protein 50. Reduced PRMT5 activity increases HPC proliferation
and promotes erythroid differentiation.
Mediated Transformation Many PV patients have a low burden of JAK2V617F, as assessed
by DNA sequencing, suggesting that there is either a subpopulation
The JAK2V617F mutation occurs within the JH2 domain of JAK2, of cells that are homozygous for JAK2V617F mixed with WT cells
which has significant homology to the kinase domain of JAK2 (JH1) or a clonal population of cells with one WT copy of JAK2 and one
but lacks catalytic activity (see Fig. 68.4). The JH2 domain exerts an mutant copy of JAK2. In PV, data from clonality or quantitative
inhibitory effect on JAK2 kinase activity, and the V617F mutation is JAK2V617F assessment and from colony assays suggest that most PV
predicted to disrupt this inhibition. In vitro kinase assays with patients have a subpopulation of cells homozygous for JAK2V617F,
JAK2V617F and WT JAK2 have revealed that JAK2V617F has greatly but in ET, clonal progenitor cells are heterozygous for JAK2V617F.
increased kinase activity, as assessed by autophosphorylation and by It is therefore important to determine whether the WT allele can
substrate phosphorylation. Ectopic expression of JAK2V617F in interfere with the ability of JAK2V617F to constitutively signal in the
either epithelial or hemopoietic cell lines results in autophosphoryla- heterozygous state and whether there is an effect of gene dosage on
tion of mutant JAK2, but not the WT JAK2, and activation of the activation of signal transduction pathways. Transient coexpression
downstream signaling events. BAF3 or FDCP cell lines expressing of WT JAK2 does not interfere with the ability of JAK2V617F to
the EPOR and engineered to stably express JAK2V617F are largely autophosphorylate even when WT JAK2 is expressed at higher levels
independent of the addition of exogenous growth factors and are than the mutant kinase. This suggests that JAK2V617F kinase activity
hypersensitive to EPO. Coexpression of JAK2V617F and a homodi- is unaffected by coexpression of WT JAK2. In contrast, when
meric type 1 cytokine receptor (EPOR, TPOR, or G-CSFR) facilitates JAK2V617F and WT JAK2 were coexpressed in Ba/F3 cells, cytokine-
the transformation of cells to growth factor independence, suggesting independent growth was attenuated, suggesting in this cellular
that the mutant JAK2 requires a receptor scaffold to be active. This context that WT JAK2 is able to interfere with JAK2V617F-mediated
contrasts with the effects of the TEL-JAK2 fusion gene, which can transformation. SOCS proteins bind to the JH1 catalytic loop and
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readily transform cells on its own, presumably because of the strong target JAK2 for degradation. SOCS-1 and SOCS-3 bind to the
homodimerization effects of the TEL moiety. Ectopic expression of catalytic groove belonging to JAK2 and inhibit its catalytic activity.
JAK2V617F can also sensitize cells to the effects of IGF1, a charac- SOCS-3 binds to EPOR and JAK2 to inhibit EPOR signaling. SOCS
teristic feature of PV progenitors. proteins inhibit JAK2 by functioning as E3 ubiquitin ligases.
Expression of JAK2V617F in hematopoietic cells activates intracel- Although SOCS-1, SOCS-2, and SOCS-3 inhibit phosphorylation
lular signaling pathways downstream of the EPOR, including STAT5, of WT JAK2, they are incapable of blocking phosphorylation of
STAT3, the MAP kinase pathway, and the PI3K–Akt pathway. JAK2V617F. On the basis of their findings, SOCS-3 appears to be
STAT5 is normally phosphorylated by the cytokine receptor–JAK2 unable to inactivate JAK2V617, and SOCS-3 itself is not degraded
complex, and phosphorylated STAT5 then translocates to the nucleus but accumulates and actually promotes the further phosphorylation
and activates the transcription of target genes. The target genes of of JAK2V617F. Such dysregulation likely enhances JAK2V617F-
STAT5 include Bcl-X L , an important antiapoptotic protein known induced cell proliferation and prolongs signaling. These data have
to be expressed in increased levels in PV proerythroblasts. The pos- been suggested as an explanation for why JAK2V617F hematopoiesis
sibility that STAT5-mediated activation of Bcl-X L is important in the predominates in PV heterozygotes. Low levels of JAK2V617F signal-
pathogenesis of PV was suggested by observations that expression of ing in JAK2V617F heterozygotes likely induce SOCS-3, which would
either constitutively active STAT-5 or Bcl-X L resulted in spontaneous downregulate JAK2 WT signaling and enhance signaling by
erythroid colony formation. Furthermore, the degree of apoptosis JAK2V617F, permitting the malignant clone to predominate.
can also be altered by p53 levels. JAK2V617F appears to function-
ally inactivate p53 by upregulating MDM2, an E3 ubiquitin ligase,
thereby decreasing the degree of apoptosis. In addition, cells express- Additional Mutations Associated With
ing JAK2V617F display constitutive activation of the MAP kinase Polycythemia Vera
pathway (as assessed by phosphorylation of ERK), and of the PI3K
pathway (as assessed by phosphorylation of AKT). Furthermore, After the application of whole-genome assays (comparative genomic
JAK2 has been shown to play a role in cellular MPL trafficking. hybridization and SNPs) as well as whole-genome sequencing, an
Decreased expression of MPL on the cell surface of platelets and Mks increasing number of mutations have been observed in patients with
is an established feature of PV and MF. Lower expression of MPL Philadelphia chromosome-negative MPNs. These mutations are not