Table S9. Genes that may mediate transformation by NUP98 fusions.
Upregulated GenesHomeobox genes / Several genes that encode homeodomain-containing transcription factors are upregulated by NUP98 fusions. These include the clustered (type I) HOX genes HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXB3, HOXB5, HOXB6, and HOXB7 as well as the non-clustered (type II) homeobox genes, MEIS1 and PBX3. Homeobox genes play important roles in normal hematopoiesis and in leukemogenesis (1, 2). Several are expressed early in hematopoiesis and downregulated with differentiation (3). Overexpression of HOXA9 in mouse bone marrow causes AML that is accelerated by coexpression of MEIS1 (4). MEIS1 accelerates the induction of AML by NUP98-HOXA9 in mice (4). Several homeobox genes including HOXA9, HOXA7, and MEIS1 are frequently overexpressed in human AML and are associated with worse prognosis (5-9). Homeobox gene overexpression is believed to mediate the leukemogenic effects of MLL fusions (10). The induction of several homeobox genes by both NUP98-HOXA9 and NUP98-DDX10 suggests that homeobox genes play an important role in leukemic transformation by NUP98 fusions. This notion is supported by data showing that homeobox genes are upregulated in murine bone marrow cells expressing other leukemogenic NUP98 fusions such as NUP98-HOXD13, NUP98-PMX1, NUP98-NSD1, and NUP98-HHEX (11-15).
EGR1 / This is an immediate-early gene that encodes a zinc finger transcription factor (16). It is upregulated in human AML with monocytic differentiation (17) and in prostate cancer (18). It is also upregulated in a mouse model of acute promyelocytic leukemia (19) and there is evidence that it mediates the proliferative effects of erythropoietin on mouse erythroblasts (20). These data suggest that upregulation of EGR1 by NUP98 fusions may contribute to the leukemic transformation of primary human cells, particularly to the increased numbers of erythroid cells observed.
SOX4 / This is a transcription factor that contains an HMG DNA-binding domain (21). It can cooperate with either MEF2C or EVI1 to induce AML in mice (22, 23) and is induced by the AML-associated RUNX1-ETO fusion oncoprotein in primary human hematopoietic cells (24).
MYCN / This is a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that is well known for its amplification and overexpression in neuroblastoma and other pediatric tumors (25). MYCN is also amplified and overexpressed in lymphomas (26, 27). More recently, it was reported that MYCN is overexpressed in many cases of pediatric AML and that its overexpression in mice leads to the development of AML (28). Therefore upregulation of MYCN may contribute to leukemic transformation by NUP98 fusions.
ZNF521 / Also known as EHZF, this is a zinc finger protein that is expressed in primitive human CD34+ hematopoietic cells and declines with differentiation (29). It is expressed in most cases of AML (29). It interacts with of SMAD1, SMAD4, and GATA-1 and regulates their transcriptional activity (29, 30). There is evidence that it inhibits erythroid differentiation by inhibiting the transcriptional activity of GATA-1 (30).
HEY1 / This is a b-HLH protein also known as HERP2 that acts as a transcriptional repressor. It is a mediator in the Notch signaling pathway (31) and is implicated in the pathogenesis of tumors of the brain, lung, pancreas, bone, skin, and kidney (32-37). Notch signaling is believed to be important in the self-renewal of hematopoietic stem cells (38) and in the pathogenesis of hematologic malignancies, particularly T-lymphoblastic leukemia (39). These data suggest that upregulation of HEY1 may contribute to the long-term proliferation and increased numbers of LTC-ICs induced by NUP98 fusions. HEY1 plays a role in erythroid differentiation: in a mouse cell line, Notch signaling associated with HEY1 upregulation increased the numbers of erythroid cells (40), and upregulation of HEY1 by JUN in primary human hematopoietic cells was associated with a block in erythroid differentiation (41). The somewhat different results obtained in these two studies may reflect the different experimental systems used, but the data nevertheless suggest that the increased numbers of erythroid cells and the disrupted erythroid differentiation that we observed in primary human cells expressing NUP98 fusions may be mediated at least in part by upregulation of HEY1.
PTGS2 / This gene encodes prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase 2), better known as cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandins G2 and H2, precursors of a number of different prostaglandins (42). COX-2 is overexpressed in many tumors resulting in overproduction of prostaglandins, including PGE2, which is thought to be important in carcinogenesis (42). Many types of cancer overexpress COX-2 and its inhibition with drugs such as aspirin can be used in the prevention and treatment of cancer (43). A number of hematopoietic malignancies, including chronic myleogenous leukemia, chronic lymphocytic leukemia, lymphomas, and myeloma, show overexpression of COX-2, which is associated with a worse prognosis (44). There is evidence that the use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a lower incidence of lymphomas and acute leukemia (45). In particular, NSAID use can reduce the risk of AML, particularly the FAB M2 subtype (AML with maturation) (46), which is the most common subtype in cases with NUP98 gene rearrangements (47). The upregulation of PTGS2 by NUP98 fusions may contribute to the worse prognosis of AML associated with NUP98 gene rearrangements, and raises the possibility that COX-2 blockade could help in the treatment of these leukemias.
REN / This gene encodes renin, a constituent of the renin-angiotensin system (RAS) that converts angiotensinogen to angiotensin I, which in turn is converted by angiotensin-converting enzyme (ACE) to angiotensin II, a regulator of blood pressure and electrolyte balance (48). In addition to this role, the RAS may play an important role in hematopoiesis and AML (49). Indeed, angiotensin II has been shown to increase the proliferation of both primitive and committed hematopoietic progenitors (50, 51). Blasts from many AML patients express renin while normal bone marrow cells do not (52-54); in addition, angiotensinogen and ACE are also expressed in bone marrow from AML patients (55). Thus it is possible that the enhanced expression of renin is a part of the mechanism for uncontrolled cell growth in some leukemias. Interestingly, drugs that inhibit the RAS system, including ACE inhibitors and losartan, have been shown to inhibit growth and induce apoptosis in AML cell lines in vitro(56). These data raise the possibility that such drugs may be useful in the treatment of AML associated with NUP98 gene rearrangements.
ANGPT1 / Angiopoietin-1 is an angiogenic growth factor that is produced by leukemic cells from AML patients (57). It is co-expressed with its receptor, Tie2 (TEK), in AML cells and is thought to act as an autocrine and paracrine factor that enhances angiogenesis and the growth of AML cells (58-60).
RYK / This is a tyrosine kinase with an aberrant catalytic domain that acts as a receptor in Wnt signaling (61). The WNT signaling pathway, particularly the Wnt3a ligand, plays an important role in maintaining hematopoietic stem cell function (62). RYK interacts with Wnt3a and mediates Wnt signaling (63). It has been identified in maturing hematopoietic cells in mice (64). Finally, RYK is overexpressed in ovarian cancer and is capable of transforming mouse fibroblasts (65, 66).
STYK1 / Also known as NOK, this is a putative protein kinase that transforms cells in vitro and promotes their tumorigenesis and metastasis in mice (67, 68). It is overexpressed in breast and lung cancer (69, 70).
TMOD1 / This gene encodes tropomodulin 1, a member of the tropomodulin family of proteins that cap the slow-growing pointed end of actin filaments (71). Tropomodulin 1 is expressed in erythroid cells (71), and its upregulation by NUP98 fusions likely reflects the increased numbers of erythroid cells observed.
IDO2 / The first step in the catabolism of tryptophan is catalyzed by two related indoleamine 2,3-dioxygenase enzymes, IDO and IDO2(72). IDO is expressed by many human tumors and by dendritic cells and is thought to facilitate the escape of tumors from immune surveillance (73). An inhibitor of indoleamine 2,3-dioxygenase enzymes, 1-methyl-tryptophan, shows antitumor activity in mice (73). Interestingly, the D-isomer of 1-methyl tryptophan, which preferentially inhibits IDO2, shows higher antitumor activity than the L-isomer, which targets IDO (74, 75).
Downregulated genes:
RAP1A / This gene a small GTPase of the Ras family that was identified as a suppressor of the transforming activity of oncogenic Ras in NIH/3T3 cells (76). It was subsequently found to potentiate the functions of Ras in some cells and to antagonize them in others (77). RAP1A functions in integrin-mediated adhesion and signaling and in the MAP kinase cascade (77). It plays various roles in the proliferation and differentiation of hematopoietic cells (78). For example, megakaryocytic, monocytic, and lymphoid differentiation of cell lines is associated with RAP1A induction (79, 80). Maturation of megakaryocytes derived from human cord blood cells is also associated with induction of RAP1A (81). Thrombopoietin-induced megakaryocytic differentiation is mediated by sustained activation of the ERK/MAPK pathway mediated by RAP1A activation and inhibition of megakaryocytic differentiation by stromal contact is associated with a block in RAP1A activation (80, 82). Finally, RAP1A is activated by erythropoietin (83), raising the possibility that it plays a role in erythroid differentiation. Based on these data, the repression of RAP1A expression by both NUP98 fusions may play a role in their inhibition of both erythroid and myeloid differentiation.
FCGR2C / This is an isoform of FCGR2, also known as CD32. It is expressed on myelomonocytic cells and is upregulated during myeloid differentiation (84-87). Its downregulation by NUP98 fusions is consistent with the observed disruption of myeloid differentiation.
CLEC7A / This gene, also known as Dectin-1, encodes a beta-glucan receptor that is expressed on myeloid cells including monocytes/macrophages, neutrophils, and eosinophils (88-90). Its repression by NUP98 fusions may reflect disruption of myeloid differentiation.
ALOX5 / This is a member of the lipoxygenase gene family that plays a role in the synthesis of leukotrienes from arachidonic acid. It is transcriptionally upregulated during myeloid differentiation (91-93). Its repression by NUP98 fusions likely reflects disrupted myeloid differentiation.
FPR3 / This is a member of the family of formyl peptide receptors; it is also known as FPRL2 and expressed in monocytes (94). Its repression by NUP98 fusions may reflect disrupted myeloid differentiation.
A2M / This gene encodes the protease inhibitor alpha-2-macroglobulin, which is induced during the maturation of monocytes to macrophages (95, 96). Its repression by NUP98 fusions may reflect disrupted myeloid differentiation.
ELA2 / This gene encodes elastase, a component of neutrophil granules (97). Its downregulation by NUP98 fusions is consistent with their disruption of myeloid differentiation.
GADD45B / Also known as MyD118, this is one of a closely related family of genes associated with terminal myeloid differentiation and involved in the induction of cell cycle arrest and apoptosis (98). Its early downregulation by NUP98 fusions may contribute to blocked differentiation and increased proliferation.
MS4A3 / This gene, also known as HTm4, belongs to a family of proteins with 4 membrane-spanning domains and is expressed predominantly in hematopoietic cells (99). It is involved in the regulation of the cell cycle in hematopoietic cells where its overexpression leads to cell cycle arrest at the G0/G1 phase (100). Repression of the myeloid oncogene AML1-ETO in cell lines and primary AML blasts results in induction of MS4A3 (101), indicating that the proliferative effects of AML1-ETO are mediated by downregulation of MS4A3.
TRIM35 / Also known as Hls5 or MAIR, this gene is thought to act as a tumor suppressor that induces apoptosis and is associated with erythroid-to-myeloid lineage switch (102-104). Its repression by NUP98 fusions may explain the skewing towards erythroid differentiation (Figure 3); it also raises the possibility that the increased numbers of cells in long-term culture (Figure 2) may be due in part to decreased apoptosis.
ZFHX3 / This gene is also known as ATBF1 and encodes two isoforms of a transcription factor that contains multiple homeodomains and zinc fingers (105, 106). It is thought to act as a tumor suppressor that is downregulated in cancers of the prostate, breast, liver, and stomach (107-110). In neuronal cells, it induces differentiation and cell cycle arrest (111). Based on these findings, it is possible that downregulation of ZFHX3 by NUP98 fusions plays a role in their ability to block differentiation and induce proliferation in hematopoietic cells.
NDRG2 / This is a member of the N-myc downregulated gene family. It is thought to act as a tumor suppressor that is downregulated in many tumors including carcinomas of the colon and thyroid, glioblastoma, and others (112-116). Its repression by NUP98 fusions suggests a similar role as a tumor suppressor in AML.
RAD18 / This gene encodes a ubiquitin ligase involved in DNA repair (117, 118). Its downregulation by NUP98 fusions may predispose cells to further DNA damage. For example in patients treated with topoisomerase II inhibitors, who are susceptible to NUP98 gene rearrangements (47), such a mechanism may lead to additional mutations that could cooperate with the NUP98 fusion in producing AML.
EED / This is one of the Polycomb Group (PcG) gene that act as transcriptional repressors (119). Two Polycomb repressive complexes (PRC) have been described: PRC1 and PRC2. EED is a component of the PRC2 complex that methylates H3K27 and represses the expression of several HOX genes (119, 120). Interestingly, several HOX genes including HOXA7, HOXA9, MEIS1, and others are upregulated by NUP98 fusions (Supplementary Tables S6 – S8). This upregulation of HOX genes may be explained at least in part by repression of EED.
GPX3 / This gene encodes glutathione peroxidase 3, a selenium dependent enzyme that plays a role in detoxifying reactive oxygen species and is downregulated in a number of cancers including those of the prostate, esophagus, and stomach (121-125). Its reression by NUP98 fusions suggests that it may play a role in AML as well.
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