New kinase-deficient PAK2 variants associated with Knobloch syndrome type 2.

The p21-activated kinase (PAK) family of proteins regulates various processes requiring dynamic cytoskeleton organization such as cell adhesion, migration, proliferation, and apoptosis. Among the six members of the protein family, PAK2 is specifically involved in apoptosis, angiogenesis, or the development of endothelial cells. We report a novel de novo heterozygous missense PAK2 variant, p.(Thr406Met), found in a newborn with clinical manifestations of Knobloch syndrome. In vitro experiments indicated that this and another reported variant, p.(Asp425Asn), result in substantially impaired protein kinase activity. Similar findings were described previously for the PAK2 p.(Glu435Lys) variant found in two siblings with proposed Knobloch syndrome type 2 (KNO2). These new variants support the association of PAK2 kinase deficiency with a second, autosomal dominant form of Knobloch syndrome: KNO2.

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

A novel germline hyperactivating JAK2 mutation L604F.

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

PAK1 and PAK2 in cell metabolism regulation.

P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. In contrast, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms.

Integrin expression and adhesivity to fibronectin in primary acute myeloid leukemia cells: Impact of NPM1 and FLT3 mutations.

OBJECTIVES: Interaction of leukemia cells with the bone marrow extracellular matrix promotes cell survival and resistance to chemotherapy. In this work, we analyzed integrin expression and adhesivity to fibronectin in primary cells from patients with acute myeloid leukemia. METHODS: Surface expression of integrins ß1 and αVß3 on primary leukemia cells (N = 46) was correlated with the stem cell marker CD34, as well as with cell adhesivity to fibronectin. The results were analyzed with regard to the mutational status of NPM1 and FLT3 genes. RESULTS: The integrin ß1 was omnipresent, whereas αVß3 was often more expressed on CD34-positive cells. In particular, higher αVß3 expression on CD34+ cells was associated with NPM1 mutation (P = .0018). Monocytic leukemias had significantly higher αVß3 expression compared to less maturated cases (P = .0008). Cells from patients with internal tandem duplications in FLT3 (FLT3-ITD) had lower adhesivity to fibronectin compared to cells with wild-type FLT3 (P = .031), specifically in less differentiated myeloblasts. Inhibition of a putative FLT3-ITD target, EZH2, increased cell adhesivity in MV4-11 cell line (P = .024). CONCLUSIONS: The integrin αVß3 is expressed in particular on CD34+ cells with NPM1 mutation and might have a prognostic value in patients with mutated NPM1. FLT3-ITD is associated with lower cell adhesivity, especially in patients with less differentiated leukemias.

High PD-L1 Expression Predicts for Worse Outcome of Leukemia Patients with Concomitant NPM1 and FLT3 Mutations.

Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).

Low-Dose Actinomycin-D Induces Redistribution of Wild-Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells.

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

Simplifying procedure for prediction of resistance risk in CML patients - Test of sensitivity to TKI ex vivo.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have dramatically improved chronic myeloid leukemia therapy. While imatinib remains to be the first line therapy, about 30% of patients develop resistance or intolerance to this drug and are recommended to switch to other TKIs. Nilotinib and dasatinib are currently implemented into the first line therapy and other inhibitors have already entered the clinical practice. This opens further questions on how to select the best TKI for each patient not only during the therapy but also at diagnosis. The individualized therapy concept requires a reliable establishment of prognosis and prediction of response to the available TKIs. We tested the ex vivo sensitivity of patient primary leukocytes to imatinib, nilotinib and dasatinib - two concentrations of each inhibitor for 48h incubation - and we evaluated the usefulness of such tests for the clinical practice. Besides reflecting the actual sensitivity to the therapy, our optimized simple tests were able to predict the outcome in 90/87% of patients, for the next 12/24months, respectively. According to these results, the presented ex vivo testing could help clinicians to select the appropriate drug for each patient at diagnosis and also at any time of the therapy.

Characterization of a new human plasma cell leukemia cell line UHKT-944.

OBJECTIVE: A new interleukin-6 (IL-6)-dependent plasma cell leukemia cell line UHKT-944 was established from bone marrow cells derived from a 55-yr-old man with plasma cell leukemia. RESULTS: The cell line possesses phenotypic characteristics of plasma cells including the production of a monoclonal immunoglobulin IgA1-kappa. VH3-9 region of IgVH genes was rearranged and somatically hypermutated. The UHKT-944 cells were found to be negative for most of tested B-cell, T-cell, and myeloid markers. According to cytogenetic analysis, the cells were classified as near tetraploid with several numerical and structural abnormalities including the t(14;20) involving IgH locus. CONCLUSION: The established permanent plasma cell leukemia cell line is a suitable model for the study of cellular and molecular mechanisms of pathogenesis of this rare malignant disease.

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease.

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30-90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na(+), while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na(+), K(+) and Cl(-) channels failed to prevent apoptosis or AVD.

NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia.

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes.

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 µM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 µM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 µM Q-VD-OPh while 10 µM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.

Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

Group I p21-activated kinases in leukemia cell adhesion to fibronectin.

P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins ß1 and αVß3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.

NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization.

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

Suberoylanilide hydroxamic acid (SAHA) at subtoxic concentrations increases the adhesivity of human leukemic cells to fibronectin.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate).

Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways.

Nucleophosmin in leukemia: Consequences of anchor loss.

Nucleophosmin (NPM), one of the most abundant nucleolar proteins, has crucial functions in ribosome biogenesis, cell cycle control, and DNA-damage repair. In human cells, NPM occurs mainly in oligomers. It functions as a chaperone, undergoes numerous interactions and forms part of many protein complexes. Although NPM role in carcinogenesis is not fully elucidated, a variety of tumor suppressor as well as oncogenic activities were described. NPM is overexpressed, fused with other proteins, or mutated in various tumor types. In the acute myeloid leukemia (AML), characteristic mutations in NPM1 gene, leading to modification of NPM C-terminus, are the most frequent genetic aberration. Although multiple mutation types of NPM are found in AML, they are all characterized by aberrant cytoplasmic localization of the mutated protein. In this review, current knowledge of the structure and function of NPM is presented in relation to its interaction network, in particular to the interaction with other nucleolar proteins and with proteins active in apoptosis. Possible molecular mechanisms of NPM mutation-driven leukemogenesis and NPM therapeutic targeting are discussed. Finally, recent findings concerning the immunogenicity of the mutated NPM and specific immunological features of AML patients with NPM mutation are summarized.

PAK1, PAK1Δ15, and PAK2: similarities, differences and mutual interactions.

P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions.

AML-associated mutation of nucleophosmin compromises its interaction with nucleolin.

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.