MLL-ENL Leukemia Research Articles

Abstract 2966: The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia

Abstract MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed >300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2966.

SIRT2 regulates proliferation and chemotherapy response of MLL-ENL-driven acute myeloid leukemia

Both MLL-AF9 and MLL-ENL leukemia fusion proteins drive oncogenic transformation of hematopoietic cells through their N-terminal DNA/histone binding mixed-lineage leukemia 1 domain and C-terminal fragment of AF9 or ENL containing an unstructured linker region and the ANC1 homology domain, which recruits transcription factors. Despite of their structural similarity, acute myeloid leukemia (AML) patients bearing MLL-ENL show more adverse outcomes compared to those with MLL-AF9. We recapitulated the clinical patterns of these two MLL-fusions driven AMLs using murine models and found that MLL-ENL AML cells showed slower cell cycle progression and more resistance to standard chemotherapy than MLL-AF9 cells. These phenotypes were primarily controlled by the linker regions of ENL and a highly conserved lysine residue K469 within. Substitution of K469 with an acetylated mimic glutamine abolished the ability of MLL-ENL to suppress proliferation and promote chemo-resistance. We showed that deacetylase Sirt2 might act as an upstream regulator of MLL-ENL. Deletion of Sirt2 promoted proliferation of AML cells with either MLL fusions. Importantly, loss of Sirt2 greatly enhanced the sensitivity of the MLL-ENL AML cells to chemo-treatment. Taken together, our study uncovered a unique regulatory role of Sirt2 in leukemogenesis and suggested targeting SIRT2 as a new way to sensitize MLL-ENL AML patience for chemotherapy.

Tip60 activates Hoxa9 and Meis1 expression through acetylation of H2A.Z, promoting MLL-AF10 and MLL-ENL acute myeloid leukemia.

Chromosome translocations involving the MLL gene are common rearrangements in leukemia. Such translocations fuse the MLL 5'-region to partner genes in frame, producing MLL-fusions that cause MLL-related leukemia. MLL-fusions activate transcription of target genes such as HoxA cluster and Meis1, but the underlying mechanisms remain to be fully elucidated. In this study, we discovered that Tip60, a MYST-type histone acetyltransferase, was required for the expression of HoxA cluster and Meis1 genes and the development of MLL-fusion leukemia. Tip60 was recruited by MLL-AF10 and MLL-ENL fusions to the Hoxa9 locus, where it acetylated H2A.Z, thereby promoting Hoxa9 gene expression. Conditional deletion of Tip60 prevented the development of MLL-AF10 and MLL-ENL leukemia, indicating that Tip60 is indispensable for the leukemogenic activity of the MLL-AF10 and MLL-ENL-fusions. Our findings provide novel insight about epigenetic regulation in the development of MLL-AF10 and MLL-ENL-fusion leukemia.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

The developmental stage of the hematopoietic niche regulates lineage in MLL-rearranged leukemia.

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

PRMT5 Inhibition Selectively Targets Acute Myeloid Leukemia Stem Cells Though a p53-Dependent Mechanism

Background: Targeting leukemic stem cells without detrimental effects on hematopoietic stem cells is a major goal for improving cure rates for acute myeloid leukemia (AML). Strategies include targeting leukemia specific mutations or pathways where leukemic cells are more dependent, leading to so-called synthetic lethality. One potential target for the latter is PRMT5, an arginine methyltransferase that methylates arginine on histones and a large number of non-histone proteins including components of the spliceosome. PRMT5 is essential for the maintenance of normal hematopoietic stem cells, through p53-dependent and independent mechanisms.Aim: To determine the relative importance of PRMT5 in normal and leukemic stem cells using genetic and pharmacologic approaches.Hypothesis: Survival of leukemic stem cells will be more dependent on PRMT5 than normal hematopoietic stem cells.Results: Using a conditional-null allele, we deleted Prmt5 in two mouse models of AML; AML1-ETO and MLL-ENL. Deletion of Prmt5 in AML1-ETO leukemia dramatically improved survival, with relapse only occurring in the setting of Prmt5-expressing cells. In contrast, deletion of Prmt5 in MLL-ENL leukemia had little effect.After screening a 350,000-compound library, we developed a potent and selective SAM-dependent inhibitor (CTx293) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. We tested the in vivo activity of CTx293 in the AML1-ETO model generated on either a p53 wild-type or p53-/- background. Twice daily administration of CTx293 for 14 days demonstrated absolute p53 dependent activity, with prolongation of mean survival from 35 to 130 days (Figure 1A).To directly compare the effects of PRMT5 inhibition on leukemic and normal progenitors, we examined the numbers of cells within the same animal during treatment with CTx293. After 3 days, there was a two-fold reduction in both leukemic and normal progenitors. However, after 7 days treatment, leukemic progenitors had reduced more than 1000-fold whilst normal progenitors (in the same mouse) had recovered (Figure 1B). To understand this differential effect on normal and leukemic progenitors, we FACS-isolated cells after 3 days therapy. While, there was evidence of p53 activation in both normal and leukemic progenitors, the downstream effects were quite distinct, with leukemic progenitors showing activation of apoptosis.We tested the potency of CTx293 on primary human AML cells using a 14-day semi-solid agar growth assay. This demonstrated greater sensitivity of most AML samples (LD<30 nM) compared with healthy CD34+ cells (LD>100 nM). Of note, TP53-mutant samples were more resistant. Finally, we demonstrate activity of single agent CTx293 in 4 patient-derived xenografts.Conclusion: We have used both genetic and pharmacologic approaches to show that PRMT5 is an attractive target for eliminating leukemic stem cells through a p53-dependent mechanism without toxicity to healthy stem cells. [Display omitted] DisclosuresToulmin:CRC Cancer Therapeutics: Research Funding. Sonderegger:CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Street:CRC Cancer Therapeutics: Employment, Patents & Royalties; MERCK: Membership on an entity's Board of Directors or advisory committees. Stupple:CRC Cancer Therapeutics: Employment. Jane:CRC Cancer Therapeutics: Patents & Royalties. Wei:Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.

Abstract 2088: PU.1 enrichment to intronic enhancers is required for meis1 gene expression in both hematopoiesis and leukemogenesis

Abstract Hematopoiesis is a tightly regulated process with multiple transcription factors (TFs) involved sequentially. Aberrant expression of the lineage specific transcription factors may disturb normal differentiation of hematopoietic cells and result in leukemia. Myeloid Ectropic viral Integration Site 1 (MEIS1), induces and maintains MLL transformation, and plays a rate-limiting regulatory role in MLL leukemogenesis. Meis1 gene is identified as the downstream targets of PU.1, another important hematopoietic transcription factor, which is inversely correlated with worse prognosis. Though multiple regulatory elements have been identified throughout the Meis1 locus, little is known about how PU.1 plays its role on Meis1 gene expression. To gain insight into the transcriptional regulatory role of PU.1 during myeloid differentiation, we isolated the hematopoietic stem/progenitor cells (HSPC, Lin-kit+CD34+ cells) and matured myeloid cells (Lin+Gr1+cells) from bone marrows of C57 mice, performed ChIP sequencing assay, and analyzed PU.1 enrichment and histone modifications. The ChIP seq data was then confirmed with an induced differentiation model of mouse MLL-ENL leukemia cells. In addition to DNaseI hypersensitivity, the PU.1 binding elements were also characterized with H3K4me1 and H3K27ac modification. We studied the transcriptional activation role of one common PU.1 binding elements with luciferase assay. Insertion of PU.1 binding elements before the promoter increased the luciferase reporter activity, suggesting that the PU.1 binding region be the enhancer element of Meis1 gene. The association between PU.1 enrichment and Meis1 gene expression was studied in further with PU.1 knocking down assay in the MLL leukemia cells. Bioinformatics analysis results suggest that there were other transcription factors, including HOXA9 and MEIS1 itself enriched around the PU.1 binding sites. Our results suggest that PU.1 enrichment to the intronic enhancers of Meis1 gene is required for the proper expression during hematopoiesis. PU.1 together with histone modification and many other TFs, including MEIS1 itself, contributed to the hematopoietic expression of Meis1 gene. (The work is supported by National Natural Science Foundation of China (Grant number 81100380) to Y.L.) Citation Format: Yajun Li, Bo Li, Qiang Li, Lixia Zhang, Ping Li. PU.1 enrichment to intronic enhancers is required for meis1 gene expression in both hematopoiesis and leukemogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2088. doi:10.1158/1538-7445.AM2015-2088

The CRM1 Nuclear Export Receptor Activates HOXA Gene Expression in Leukemogenesis

HOXA genes are effectors of oncogenic transformation that are frequently upregulated in myeloid and T-cell acute leukemias. Chromosomal translocation-derived oncoproteins, including MLL fusions, NUP (NUP98 or NUP214) fusions or CALM-AF10, bind to HOXA genes and result in their overexpression. We have previously demonstrated that a CRM1-dependent Nuclear Export Signal (NES) within CALM is essential for CALM-AF10’s ability to upregulate HOXA genes and cause leukemia in mice. Interfering with the CRM1/CALM-AF10 interaction by either genetic or pharmacologic inhibition abolishes CALM-AF10’s ability to bind to and activate HOXA gene expression. Furthermore, we showed that CRM1 binds to HOXA loci, suggesting that CRM1 recruits CALM-AF10 to its target genes.To explore whether CRM1 is also involved in the upregulation of Hoxa genes associated with MLL- and NUP98-fusion genes, we measured Hoxa transcript levels in murine leukemia cells treated with the CRM1 inhibitor Leptomycin B (LMB). LMB is a small molecule that covalently binds to the NES binding domain of CRM1 and blocks its ability to interact with NES partner proteins. We found that treatment of MLL-AF10, MLL-ENL, NUP98-HOXA9 or NUP98-AF10 leukemia cells with LMB (1 nM, 2 hours) causes a 50% reduction of Hoxa7, Hoxa9, Hoxa10 and Hoxa11 levels, similar to what is observed in CALM-AF10 leukemia cells. This suggests that in addition to its ability to interact with CALM-AF10, CRM1 may also participate in the transcriptional activation of Hoxa genes caused by MLL- or NUP98-fusion proteins.To demonstrate the importance of the CRM1/CALM interaction in CALM-AF10-dependent oncogenesis, we studied the biological activity of an artificial CRM1-AF10 fusion protein. Using a murine bone marrow clonogenic progenitor replating assay, we found that while native CRM1 overexpression did not result in transformation, the CRM1-AF10 fusion significantly increased the self-renewal of clonogenic progenitors. This effect was even more pronounced when CRM1 was fused to the MLL partner ENL: transduction with a CRM1-ENL fusion gene caused the immortalization of clonogenic bone marrow progenitors. Both CRM1-AF10- and CRM1-ENL-transduced progenitors displayed overexpression of Hoxa genes. To investigate the leukemogenic potential of CRM1-AF10in vivo, we transplanted mice with retrovirally transduced bone marrow progenitors and found that CRM1-AF10 induces myeloid neoplasms with a low penetrance and long latency (after more than a year of observation, 5 of 15 mice developed myeloid neoplasms between 160 and 220 days). These primary CRM1-AF10 leukemias could be transplanted to secondary recipients and cause myeloid leukemias with a shorter latency. Experiments to determine the leukemogenic potential of CRM1-ENLin vivo are ongoing. In contrast to CRM1-AF10, CRM1-ENL-transduced progenitors displayed a marked proliferative advantage in all transplanted mice (assessed by the elevation in the percentage of GFP-expressing CRM1-ENL-transduced cells in the peripheral blood over time); mice transplanted 74 days ago will be followed to determine survival curves.In summary, our results demonstrate that CRM1 regulates the expression of Hoxa genes in mouse leukemia cells, and alteration of CRM1’s activity can drive murine leukemogenesis. This has implications for understanding the mechanisms of HOXA deregulation in human leukemias induced by various fusion oncoproteins. It is noteworthy that in addition to interacting directly with CALM-AF10 through the NES, CRM1 physiologically interacts with NUP98 and NUP214 to facilitate transport through the nuclear pore. Our data also suggest that the anti-tumor effects of CRM1 inhibitors (Selective Inhibitors of Nuclear Export, SINEs) currently undergoing clinical trials, could be mediated, at least in part, by their ability to block the transcriptional activation of tumor-promoting genes by CRM1. DisclosuresNo relevant conflicts of interest to declare.

Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia

FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wild-type (wt) FLT3, which is highly expressed in many hematologic malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins, deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210(BCR-ABL)-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 invivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3(+) compared to Flt3(-) wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells invitro by MLL-ENL and does not affect colony formation by MLL-ENL LSCs invitro, suggesting that invitro models do not reflect the invivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation invivo, which is, however, not universal.

Flt3 Does Not Play a Critical Role in Murine Myeloid Leukemias Induced by MLL Fusion Genes

Leukemias harboring MLL translocations are frequent in children and adults, and respond poorly to therapies. The receptor tyrosine kinase FLT3 is highly expressed in these leukemias. In vitro studies have shown that pediatric MLL-rearranged ALL cells are sensitive to FLT3 inhibitors and clinical trials are ongoing to measure their therapeutic efficacy. We sought to determine the contribution of Flt3 in the pathogenesis of MLL-rearranged leukemias using a myeloid leukemia mouse model. Bone marrow from Flt3 null mice transduced with MLL-ENL or MLL-CBP was transplanted into host mice and Flt3 −/− leukemias were compared to their Flt3 wild type counterparts. Flt3 deficiency did not delay disease onset and had minimal impact on leukemia characteristics. To determine the anti-leukemic effect of FLT3 inhibition we studied the sensitivity of MLL-ENL leukemia cells to the FLT3 inhibitor PKC412 ex vivo. As previously reported for human MLL-rearranged leukemias, murine MLL-ENL leukemia cells with higher Flt3 levels were more sensitive to the cytotoxicity of PKC412. Interestingly, Flt3 deficient leukemia samples also displayed some sensitivity to PKC412. Our findings demonstrate that myeloid leukemias induced by MLL-rearranged genes are not dependent upon Flt3 signaling. They also highlight the discrepancy between the sensitivity of cells to Flt3 inhibition in vitro and the lack of contribution of Flt3 to the pathogenesis of MLL-rearranged leukemias in vivo.

MLL-ENL leukemia burden initiated in femoral diaphysis and preceded by mature B-cell depletion

Molecular and cellular events that resulted in leukemia development are well characterized but initial engraftment and proliferation of leukemic cells in bone marrow and early modifications of the bone marrow microenvironment induced by engrafted leukemic cells remain to be clarified. After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia fusion protein in non-conditioned syngenic mice, kinetics of leukemic burden and alterations of femoral hematopoietic populations were followed using an in vivo confocal imaging system and flow cytometry. Three days after injection, 5% of leukemic cells were found in femurs. Little proliferation of engrafted leukemic cells could then be detected for more than two weeks while the number of femoral leukemic cells remained stable. Twenty days after injection, leukemic cells preferentially proliferated in femoral diaphysis where they formed clusters on the surface of blood vessels and bone. B220(+) lymphoid cells were found near these leukemic cell clusters and this association is correlated with a decreased number of femoral B220(+)IgM(+) cells. Increasing the number of injected leukemic cells or conditioning recipient mice with γ-irradiation resulted in leukemic cell development in diaphysis and knee. Competition experiments indicate that proliferation but not engraftment is a rate-limiting factor of leukemic cells spreading in diaphysis. Finally, 30 days after injection leukemia developed. After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia into syngenic mice, leukemic cell burden preferentially initiates in femoral diaphysis and is preceded by changes of femoral B-lymphoid populations.

Loss of AML1/Runx1 accelerates the development of MLL-ENL leukemia through down-regulation of p19ARF

AbstractDysfunction of AML1/Runx1, a transcription factor, plays a crucial role in the development of many types of leukemia. Additional events are often required for AML1 dysfunction to induce full-blown leukemia; however, a mechanistic basis of their cooperation is still elusive. Here, we investigated the effect of AML1 deficiency on the development of MLL-ENL leukemia in mice. Aml1 excised bone marrow cells lead to MLL-ENL leukemia with shorter duration than Aml1 intact cells in vivo. Although the number of MLL-ENL leukemia-initiating cells is not affected by loss of AML1, the proliferation of leukemic cells is enhanced in Aml1-excised MLL-ENL leukemic mice. We found that the enhanced proliferation is the result of repression of p19ARF that is directly regulated by AML1 in MLL-ENL leukemic cells. We also found that down-regulation of p19ARF induces the accelerated onset of MLL-ENL leukemia, suggesting that p19ARF is a major target of AML1 in MLL-ENL leukemia. These results provide a new insight into a role for AML1 in the progression of leukemia.

Consistent Deregulation of Gene Expression between Human and Murine MLL Rearrangement Leukemias

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Identification of Genes Deregulated in Both Human and Murine MLLRearrangement Leukemias.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. The mixed lineage leukemia (MLL) gene was identified as a common target of chromosomal translocations associated with human acute leukemias; it is located on human chromosome 11 band q23 and on mouse chromosome 9. More than 50 different loci are rearranged in11q23 leukemias involving MLL, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In general, MLL rearrangements are associated with a poor prognosis. MLL-ELL and MLL-ENL resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3) respectively are two common examples of these rearrangements. These two fusions are frequently involved in human AML, while MLL-ENL is also involved in human ALL. There is a common observation that important biological properties are often conserved across species. Cross-species sequence comparison has been widely used to infer gene function, but it is becoming apparent that sequence similarity is not always proportional to functional similarity. To determine the function of a gene precisely, therefore, we need to investigate not only its sequence characteristics but also its expression characteristics. Model organisms have contributed substantially to our understanding of the etiology of human disease and the development of new treatment methodologies. However, although genetically engineered mouse leukemia models have been established for many years, there are few systematic studies to identify and study the genes that exhibit similar abnormal expression patterns in both human leukemia and mouse leukemia model cells. To perform an interspecies gene expression comparative study in leukemia, we used the serial analysis of gene expression (SAGE) technique to examine gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both humans and mice. We obtained 484,303 total SAGE tags for the nine samples and a total of 103,899 unique SAGE tags from five human and 60,993 from four mouse samples. We identified 88 genes that appeared to be significantly deregulated (32 up- and 56 down-regulated) in both human and murine MLL-ELL and/or MLL-ENL leukemia. Fifty-seven genes have not been reported previously. A large-scale quantitative real-time PCR (qPCR) assay was performed to validate the candidate genes, and 84% (36/43) of the tested SAGE candidate genes were confirmed. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9 and HOXA10) and a HOX cofactor MEIS1; their overexpression is a hallmark of MLL-rearrangement leukemia. The top down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI and CYBB. Remarkably, up-regulated genes have a much higher percentage of enrichment in Gene Ontology (GO) terms related to gene expression and transcription, whereas down-regulated genes are more enriched in GO terms related to apoptosis, signal transduction and response. Thus, the up-regulation of genes responsible for gene expression and transcription but down-regulation of genes responsible for apoptosis, signal transduction and response, can promote cell proliferation and inhibit apoptosis, and thereby contribute to the development of leukemia. We showed that the CpG islands of several significantly down-regulated genes including LIF, TGFBI and G0S2 are hypermethylated. We also examined the expression of microRNAs from the mir-17–92 cluster, which are overexpressed in human MLL-rearrangement leukemias, and showed that seven individual microRNAs (i.e., miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92) within this cluster are also overexpressed in mouse MLL-rearrangement leukemia cells. Nineteen putative targets (i.e., APP, RASSF2, SH3BP5, DBN1, ELK3, FLT1, GNAI1, HIF1A, ITGA6, MN1, POU4F1, RB1, RGL1, RNF167, SASH1, SLC24A3, TNFRSF21, WWP1 and YES1) of these microRNAs were reported and/or confirmed by our qPCR to be down-regulated in MLL-rearrangement leukemias. We further confirmed both APP and RASSF2 as direct targets of miR-17 through luciferase reporter and mutagenesis assay. The identification and validation of gene expression changes in MLL-rearrangement human and murine leukemia provides important insights into the genetic pathways that are important for MLL fusion-induced leukemogenesis.

Identification of Genes Abnormally Expressed in Both Human and Murine MLL-ELL and/or MLL-ENL Leukemia.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. Their abnormally expressed genes identify specific markers for their clinical diagnosis. Important biological properties are often conserved across species. However, although genetically engineered mouse leukemia models are well-established, few systematic studies have validated the genes that exhibit similar abnormal expression patterns in both human and mouse leukemia models. MLL-ELL and MLL-ENL fusion genes resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3), respectively, are frequently involved in human acute leukemia, and in retrovirus-mediated mouse leukemia models. We used the SAGE technique to compare gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both human and mouse. We analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL or MLL-ENL fusions, and a leukemia cell line with an MLL-ELL fusion. 484,303 SAGE tags were identified from the nine samples, yielding 103,899 unique tags in human and 60,993 in mouse samples. We identified 40 candidate genes that appear to be abnormally expressed in both human and murine MLL-ELL leukemias (2 up- and 38 down-regulated), and 72 in both human and murine MLL-ENL leukemias (23 up and 49 down). 25 candidate genes are down-regulated in both types of leukemias, and many of them can bind with and/or regulate other candidate genes in the candidate list. For example, LCN2 can bind directly with and positively regulate MMP9; MMP9 and TMSB4X may positively regulate FOS; FOS and JUNB can bind directly and positively regulate each other. JUNB may inhibit proliferation and promote apoptosis, and it was reported that inactivation of JunB in LT-HSC leads to MPD while its inactivation in committed myeloid progenitors also predisposes to leukemia evolution. LCN2 may also positively regulate apoptosis. Meanwhile, some important candidate genes are observed only in one type of leukemia. For example, both PXN and ARHGEF1 are down-regulated only in MLL-ELL leukemias. PXN can bind directly with ARHGEF1, and the latter may inhibit proliferation. Similarly, MYB is significantly upregulated only in MLL-ENL leukemias, which was reported to play a role in MLL-ENL-mediated transformation. Taken together, some common pathways may exist in the development of both types of leukemias, whereas each may also have their own pathway. The deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes, and are studying the functions and pathways of the validated candidate genes. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute myeloid leukemia, which may lead to more effective therapy for these leukemias.