Blood Cells, Molecules and Diseases 50 (2013) 227–231

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Blood Cells, Molecules and Diseases

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p15Ink4b Functions in determining hematopoietic cell fates: Implications for its roleas a tumor suppressor

Linda Wolff ⁎, Juraj Bies ⁎Laboratory of Cellular Oncology, National Cancer Institute, Room 4124, 37 Convent Dr. Bethesda, MD 20892, USA

⁎ Corresponding authors.E-mail addresses: wolffl@mail.nih.gov (L. Wolff), bie

1079-9796/$ – see front matter. Published by Elsevier Ihttp://dx.doi.org/10.1016/j.bcmd.2013.01.006

a b s t r a c t

a r t i c l e i n f o

Article history:Submitted 19 December 2012Available online 9 February 2013

(Communicated by M.A. S. Weissman, M.D.,10 January 2013)

Keywords:LeukemiaTumor suppressorHematopoiesisMethylationImmune surveillanceErythropoiesis

The p15Ink4b gene is frequently hypermethylated in myeloid neoplasia and has been demonstrated to be atumor suppressor. Since it is a member of the INK4b family of cyclin-dependent kinase inhibitors, it was ini-tially presumed that its loss in leukemic blasts caused a dysregulation of the cell cycle. However, animalmodel experiments over the last several years have produced a very different picture of how p15Ink4b func-tions in hematopoietic cells and how its loss contributes to myelodysplastic syndrome and myeloid leukemia.It is clear now, that in early hematopoietic progenitors, p15Ink4b functions outside of its canonical role asa cell cycle inhibitor. Its functions are involved in signal transduction and influence the development of ery-throid, monocytic and dendritic cells.

Published by Elsevier Inc.

p15INK4b deletion in human cancers

P15INK4b is a member of the INK4 family of cyclin-dependentkinase inhibitors. The gene, p15INK4b, also known as CDKN2b andMTS2, is located on chromosome 9q21 and is often deleted in conjunc-tion with sequences encoding two other gene products p16INK4aand p14ARF [1–3]. This has been shown to occur in lymphomas aswell as carcinomas and sarcomas [4–7]. P15INK4b and p16INK4a areboth capable of inducing cell cycle arrest in G1 by inhibiting cyclin-dependent kinases 4 and 6 (cdk4/6), whereas p14ARF is an activatorof TRP53 [2,8]. p15INK4b probably has an important backup functionfor p16INK4a in senescencewhichmay explainwhy the genes encodingboth are often deleted in human tumors. Experiments in mice showthat deletions prohibiting expression of all three INK-ARF proteinspromotes a wider spectrum of tumors than deletions affecting onlyp16Ink4a and p14ARF [9].

p15INK4b methylation in human MDS and AML

Loss of p16INK4a and p15INK4b expression in hematopoietic neo-plasms also occurs through DNA methylation of the genes encodingthese proteins [4,10,11]. Importantly, however, p15INK4b is the onlyINK4 family genemember that ismethylated inMDS andAML suggestinga specific function in the myeloid lineage. This gene is hypermethylatedin 50–60% of patients with myelodysplastic syndrome and 70–80% of

sj@mail.nih.gov (J. Bies).

nc.

patientswith AML [4]. Aberrant p15INK4bmethylation has been general-ly associated with poor prognosis in AML [12,13] and in MDS patients itsmethylation is associated with increased risk of transformation to AML[14,15]. p15INK4b hypermethylation is found in almost all FAB subtypes,although higher frequencies are seen inM1, M2, M3, andM4 [12,16–18].In patients with therapy-induced AML (t-AML) the frequency of methyl-ation is over 90% [13]. The aberrant hypermethylation is found in the CpGislands extending throughout the promoter region, exon1 and part ofintron 1 and is associated with signatures of polycomb repression andreduced H3K4 trimethylation [11,19]. A study that looked at whethermethylation occurred in all the common cytogenetic subtypes foundthat a strikingly low level of methylation occurred in AML with inv(16)[20]. This led to the finding that the inv(16)-encoded CBFβ-SMMHCsilenced p15INK4b by displacing the transcription factor RUNX1 fromthe promoter.

Demonstration of tumor suppressor function in murine models

A mouse model was developed to define a role for p15INK4b as atumor suppressor. p15Ink4b(−/−) mice, in which the second codingexon of the gene was eliminated by homologous recombination [21].These mice showed no increased susceptibility to AML over wild-typemice although the mice had extramedullary hematopoiesis and a lowincidence of angiosarcomas. Furthermore, the p15Ink4b-deficient back-ground in transgenic mice expressing the Rgr oncogene under a CD4promoter significantly decreased the incidence of thymic lymphomaswhen comparedwith CD4-Rgr progenywithwild-type or heterozygousexpression of p15Ink4b [22].

228 L. Wolff, J. Bies / Blood Cells, Molecules and Diseases 50 (2013) 227–231

Although these experiments failed to show that loss of p15Ink4bdirectly results in AML, there was reason to believe that p15Ink4bwas a tumor suppressor for AML in mice. First, retrovirus-inducedleukemias of the myelomonocytic phentype were found to have un-dergone hypermethylation of the 5′ CpG island of the p15Ink4b gene.In addition, when the knockout was redeveloped on a 129/sv back-ground using the same target vector as Latres et al. [21], it was pos-sible to show that loss of p15INK4b results in increased numbers ofmyeloid progenitors in the bone marrow. Furthermore, bone marrowfrom these mice had a competitive advantage inmyeloid cell formationin a repopulating assay [23,24].

The mouse model that has been most informative as to p15Ink4b'srole as a tumor suppressor for myeloid malignancy is a conditional onein which p15Ink4b is knocked out specifically in myeloid cells [25]. Forthis model, a floxed exon2 was introduced and the mice were crossedwith transgenic mice expressing Cre under the control of a LysM pro-moter that ensured a myeloid cell-specific expression pattern [26].These mice, called p15Ink4bfl/flLysMcre, develop monocytosis withincreased circulating monocytes in the blood starting at 5 to 7 monthsof age, but have normal levels of neutrophils, lymphocytes, plateletsand red blood cells. In addition, an expansion of myelomonocyticcells in the bone marrow was observed in these mice as evidenceby increases in mature myeloid (Gr-1+/Mac-1+) and monocytic cells(Gr-1lo/Mac-1+) as well as immature myeloid cells (Mac-1+/cKit+).Immunohistochemical staining of spleen tissue revealed a small in-crease in myelomonocytic cells in the red pulp which was confirmedby FACS analysis. A small number of mice spontaneously developeda myeloproliferative neoplasm characterized as the advanced formof chronic myelomonocytic leukemia with a significantly increasednumber of cKit+ immature cells in bone marrow and peripheral blood[25]. Since the p15Ink4bfl/flLysMcre mice did not develop acute disease,they were subjected to retroviral insertional mutagenesis to determinewhether they had increased susceptibility to acute leukemia whenprovided with additional oncogenic hits. Mice were inoculated shortlyafter birth with MOL4070LTR retrovirus [27] and monitored for15 months for disease development. Control mice developed leukemiawith low penetrance, whereas the incidence of retrovirus-inducedleukemiawas statistically highly significant for the p15Ink4bfl/flLysMcremice. Furthermore, there was a much higher incidence of myeloidtumors, mostly monocytic and myelomonocytic in p15Ink4b

fl/flLysMcre

(92%) and p15Ink4bfl/wtLysMcre (79%) mice than in control,p15Ink4bwt/wtLysMcre mice, where there was an equal distribution ofmyeloid and lymphoid leukemia. This model has, therefore, providedevidence that loss of p15Ink4b can itself promote preleukemic condi-tions and demonstrated experimentally that this gene is a tumorsuppressor for AML [25].

p15Ink4b and erythroid/myeloid cell fate

Since p15Ink4b is the only member of the INK4 family that isinactivated by DNA methylation in AML, it is of interest to determineif there is any other function specific to the myeloid lineage that canbe assigned to this gene. Initial investigations of hematopoiesis inInk4b−/− mice showed that these mice have greater numbers ofbi-potent granulocyte–macrophage progenitors (GMP) and this char-acteristic was found, in competitive repopulating studies in vivo, to beintrinsic to the cells. Interestingly, Ink4b-deficient GMPs did not cyclemore frequently thanwild-type progenitors and showed no differencesin apoptosis or self-renewal potential. However, Ink4b was shown toaffect differentiation of common myeloid progenitor (CMP) cells to-ward GMPs, resulting in an imbalance of down-stream progenitors. Invitro analysis of progenitor cells from knock-out mice demonstratedthat loss of p15Ink4b causes increased differentiation toward GMPsand decrease in differentiation toward megakaryocyte–erythroid pro-genitors (MEP) [23,28].

Based upon the data obtained from the knockout mice, it was hy-pothesized that p15Ink4b is required for efficient erythropoiesis. Sub-sequently, p15Ink4b was shown to promote erythroid differentiationand suppresses myeloid differentiation of hematopoietic progenitorsunder both normal and stress conditions. First, it was demonstratedthat p15Ink4b is expressed more highly in committed MEPs thanGMPs. More importantly, mice lacking p15Ink4b have lower numbersof primitive red cell progenitors and a severely impaired response to in-duced hematopoietic stress caused by 5-fluorouracil or phenylhydrazine.When p15Ink4b was re-introduced into the bone marrow progenitorsfromp15Ink4b−/−mice,whichhad a lowerythroid tomyeloid balance,it corrected the observed skewing in hematopoietic cell differentiation.In support of a new function for this gene outside its canonical functionas a CDKI, evidencewas provide that pRBwas not required for alterationsin the balance of myeloid/erythroid cells in p15Ink4b−/− mice. Whenp15Ink4b was introduced into a multi-potential progenitor cell line(EML,) it produced changes at the molecular level, including activationof MEK/ERK signaling, increased GATA-1, EpoR, and decreased PU.1and GATA-2 expression. These changes rendered cells more permissiveto erythroid commitment and less permissive to myeloid commitment,as demonstrated by an increase in early burst forming unit erythroid(BFU-E) formation with a concomitant decrease in myeloid progenitors[29]. Therefore, p15Ink4b functions in erythropoiesis, by maintainingproper lineage commitment of progenitors and assisting in rapid redblood cell replenishment following stress.

Role of p15Ink4b in dendritic cell development

Recently, another cell fate function of p15Ink4b was found in driv-ing dendritic cells (DCs) maturation. It was shown that mice lacking afunctional p15Ink4b in the myeloid lineage had a significantly re-duced numbers of common DC progenitor cells (CDPs) [30] that cangive rise to all DCs (Fig. 1). Concomitantly, numbers of conventional(classical) dendritic cells (cDCs), but not plasmacytoid dendritic cells(pDCs), were significantly decreased in the spleen [31]. In vitro experi-ments with bone marrow-derived DCs (BM-DCs) from hematopoieticprogenitors lacking p15Ink4b cultured in granulocytes-macrophagescolony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) confirmeda decreased capacity of myeloid progenitors lacking p15Ink4b to differ-entiate into DCs. Furthermore, the maturity of LPS-activated BM-DCsfrom knockout mice was also significantly compromised as comparedto wt-derived BM-DCs. In accordance with these data, p15Ink4b-deficient BM-DCs expressed significantly lower levels of cell surfacemolecules MHCI, MHCII and co-stimulatory molecules CD80, CD86known to be necessary for an efficient stimulation of naive T-cells [31].The immaturity of cDCs from knockout mice was functionally con-firmed by their limited ability to stimulate allogeneic T cells. Impor-tantly, enforced expression of p15Ink4b restored differentiation andmaturation defects in DCs derived frommicewith silenced endogenousp15Ink4b, confirming a positive role for p15Ink4b in development andmaturation of cDCs.

These defects in development and maturation of cDCs in the ab-sence p15Ink4b also have important implications regarding its roleas a tumor suppressor for AML, because pre-leukemic myeloid cellsdifferentiating into cDCs may have an impaired ability to providecompetent immune surveillance. DCs are the critical cells that initiateimmune surveillance and at the same time maintain appropriateself-tolerance. The scheme of dendritopoiesis suggests that myeloidprecursors of CDPs include pre-leukemic/leukemic cells with acquiredgenetic/epigenetic changes. These abnormalities that include silencingof p15Ink4b expression are required for transformation of hemato-poietic cells into leukemic cells and also they affect immune systemthrough modulation of development and/or maturation of cDCs [31].

Insight into how p15Ink4b might control maturation at the molec-ular level has been obtained. p15Ink4b potentiates transcriptionalactivity of the transcription factor PU.1, an important downstream

MPP CMP GMP

MEP

cDC

pDC

MDP

CDP

Mo-DC

CLP

MoMφφ

ErythrocytesMegakaryocytes

Lymphocytes

Granulocytes

HSC

Immature cDCs

Fig. 1. Impaired homeostasis of myeloid cells in p15Ink4b-deficient mice. A simplified scheme of hematopoietic stem cells (HSC) differentiating toward myeloid lineages ispresented. Increased production (red arrow pointing up) of granulocyte–macrophage progenitors (GMP) and monocytes (Mo), as well as decreased production (black arrowpointing down) of megakaryocyte–erythrocyte progenitors (MEP), common DC progenitors (CDP), and conventional DCs (cDC) in p15Ink4-deficient mice are indicated.Multipotent progenitors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP), macrophage/dendritic cell progenitors (MDP), monocyte-derivedDCs (Mo-DC), macrophages (Mϕ), plasmacytoid DCs (pDC).

229L. Wolff, J. Bies / Blood Cells, Molecules and Diseases 50 (2013) 227–231

target that directly regulates differentiation and maturation of cDCs,through increased phosphorylation of ERK kinases [31]. Interestingly,PU.1 is absolutely critical for development of not only cDCs, but alsoplasmacytoid DCs (pDCs). However, higher activity of this transcriptionfactor is required for development of cDCs than pDCs [32,33], whichis in a good agreement that p15Ink4b regulates mainly development

Silencing of p15Ink4b

Impaired SErythrop

Anem

Defective cDCWeak Immune A

Respo

Fig. 2. Silencing of p15Ink4b in myeloid cells affects development of several hematopoieticliferative neoplasm), CMML (chronic myelomonocytic leukemia), AML (acute myeloid leuk

of cDCs, but not pDCs. Increased expression of many Pu.1 targetsgenes, such as CD80, CD86 [34], CD40 [35] and MHCII [36,37] inBM-DCs with restored p15Ink4b confirmed further that this tumorsuppressor regulates maturation of cDCs through the transcriptionfactor PU.1. Since expression of p15Ink4b is also activated by PU.1 inmyeloid cells [38], and p15Ink4b positively regulates PU.1 [31], it is

tressed oiesisia

MonocytosisMPN (CMML-like)

Predisposition to AML

Maturationnti-Leukemicnse

cell lineages that may cumulatively contribute to AML development. MPN (myelopro-emia), cDC (conventional dendritic cells).

230 L. Wolff, J. Bies / Blood Cells, Molecules and Diseases 50 (2013) 227–231

hypothesized that this mutual cooperation creates a positive feed-back loop that amplifies differentiation and maturation of cDCs.

Concluding remarks

Experiments in animal model systems with embryonal and condi-tional knockout of p15Ink4b in mice reveal that, in addition to its ca-nonical function as regulator of cell cycle, p15Ink4b has an importantcell cycle-independent role in regulation of myeloid cell differentia-tion. It was shown that its expression in early myeloid progenitorscontributes to proper homeostatic development of monocytic, ery-throid, and dendritic cells. In early stages ofmyeloid cell differentiation,p15Ink4b modulates activities of specific instructive transcription fac-tors such as PU.1 [33,39], GATA-1, and GATA-2 [40] that drive myeloidprogenitors into specific cell lineages. Thus silencing of p15Ink4b byDNA methylation in early myeloid lineage, as observed frequently inpre-leukemic and leukemic stages of AML, has multiple consequencesthat contribute to leukemia development (summarized in Fig. 2). Thissuggests that loss of p15Ink4b expression not only favors productionof immature myeloid cells that are targets of additional mutations dur-ing leukemogenesis, but also impairs the differentiation andmaturationof cDCs from these precursors, whichmay have a negative effect on theimmune system to recognize and clear cancerous cells. In support of thishypothesis, immature, tolerogenic DCs are frequently detected in pa-tients with MDS and AML [41–43]. Since DCs modulate the nature andintensity of the adaptive immune response, they provide an attractivetarget for cancer immunotherapy. Therefore, successful targeting ofp15Ink4b re-expression in clinical treatment regimensmay not only re-store control over the of cancer cell production, but it may also improvethe anti-leukemic function of the immune system through generationof more mature, immunostimulatory DCs that prime naive T-cells torecognize and remove leukemic cells. These recent data may have alsoa translational implication in improved patient-specific anti-leukemicDCs immunotherapy to fight minimal residual disease that still remainsa critical obstacle in the successful treatment of AML.

Acknowledgments

This work was supported by the intramural research program ofthe National Cancer Institute, Center for Cancer Research, NIH

Author's contributions

L.W. and J.B. wrote the article.

Disclosure

The authors declare no conflict of interest. All authors have ap-proved the final article.

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