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Montagnards, an indigenous multitribal refugee-origin population concentrated in North Carolina, remain an invisible, medically underserved, and socioeconomically underrepresented Asian American sub-group. Yet this group is resilient, with language diversity, rich cultural traditions and family caregiving in multigenerational households. Using community-based participatory research methods, we developed and administered a two-part survey to 144 Montagnard adults, documenting socioeconomic characteristics, health indicators and lifestyle behaviors. Forty-one percent of participants had no formal education, 76% had little/no English proficiency and 28% described having a very hard time paying their bills. Seventy-seven percent were overweight per BMI category, 79% had elevated blood pressure and 100% scored high for significant depressive symptoms. Participants reported high levels of physical inactivity and daily dietary intake of MSG. However, Montagnards reported limited tobacco and alcohol use, a diet of fresh vegetables and rice, and regular church attendance. These represent protective lifestyle behaviors and targets for culturally responsive health interventions.

Creation of an inclusive environment requires a culture of equity and justice, value and respect for diverse backgrounds, and opportunities for students to engage with communities while addressing issues in science and society. These tasks are particularly challenging for institutions lacking a diverse population. Here, we demonstrate evidence of a successful model for creating an inclusive environment in an interinstitutional course between a large, public, historically black institution and a small, private primarily white institution. Because many individuals from underrepresented minority groups tend to value communal goals of working together and helping their communities, we incorporated two high-impact practices of community-engaged learning (CEL) and course-based undergraduate research experiences (CUREs) focused on health disparities research in neighboring communities. Although the research projects varied each semester, they were linked by their impact on and engagement with the community. Students practiced cultural competency skills in both small group projects within the class and engagement activities in the community. We measured the efficacy of CURE components (novel authentic research, scientific process skills, iteration, collaboration, and broader impact) through a combination of direct and indirect assessments, quantitative and qualitative analysis. More than simply scientific skills, students from both institutions developed lasting interest in working with diverse populations as well as respecting and valuing different backgrounds. This inclusive environment, combined with increased interest in research suggests that this course could potentially serve as a model for interinstitutional collaborations in creating inclusive environments that support the future success of diverse students, eventually changing the STEM research culture. Creation of an inclusive environment requires a culture of equity and justice, value and respect for diverse backgrounds, and opportunities for students to engage with communities while addressing issues in science and society. These tasks are particularly challenging for institutions lacking a diverse population. Here, we demonstrate evidence of a successful model for creating an inclusive environment in an interinstitutional course between a large, public, historically black institution and a small, private primarily white institution. Because many individuals from underrepresented minority groups tend to value communal goals of working together and helping their communities, we incorporated two high-impact practices of community-engaged learning (CEL) and course-based undergraduate research experiences (CUREs) focused on health disparities research in neighboring communities. Although the research projects varied each semester, they were linked by their impact on and engagement with the community. Students practiced cultural competency skills in both small group projects within the class and engagement activities in the community. We measured the efficacy of CURE components (novel authentic research, scientific process skills, iteration, collaboration, and broader impact) through a combination of direct and indirect assessments, quantitative and qualitative analysis. More than simply scientific skills, students from both institutions developed lasting interest in working with diverse populations as well as respecting and valuing different backgrounds. This inclusive environment, combined with increased interest in research suggests that this course could potentially serve as a model for interinstitutional collaborations in creating inclusive environments that support the future success of diverse students, eventually changing the STEM research culture.

BACKGROUND Montagnard refugees, an indigenous multilingual tribal people from Vietnam, experience lifestyle changes and post-resettlement challenges in the United States that contribute to chronic health conditions. Foundational research and health data are lacking. OBJECTIVES We describe the Montagnard Hypertension Study, a community-based participatory research (CBPR) project documenting chronic disease risk. METHODS We developed a Montagnard dictionary of hypertension-specific terminology and conducted two focus group discussions (FGD), 131 biological assessments (blood pressure, height, weight, waist circumference, scalp hair and saliva sample collection), and 127 behavioral surveys. We implemented two health fairs that offered services to the community. LESSONS LEARNED This is the first study to examine chronic disease using a CBPR framework for Montagnard health. We highlight lessons learned specific to constituents and their capacities, historical and current conflicts, and the iterative processes in CBPR design. CONCLUSIONS CBPR is a practically achievable approach to studying chronic disease risk within indigenous, tribal communities, with implications for future research with Asian American subgroups and other minority populations.

As the US transitions to a majority–minority population, the underrepresentation in the science, technology, engineering, and mathematics (STEM) workforce must be resolved to ensure that our nation maintains its competitiveness and global economic advantage. The persistent problem of retaining underrepresented minority (URM) students in STEM continues to be a national priority after several decades of attention. The role of historically black colleges and universities (HBCUs) in addressing this challenge cannot be overstated, given their history in producing African American STEM graduates. As the largest HBCU in the country, North Carolina A&T State University (NC A&T) serves a combined undergraduate and graduate population of 11,877 students, 78% of which self-identify as African American. To overcome the multiple challenges that impede retention and persistence to degree completion in biology, the Department of Biology at NC A&T has adopted a major cultural shift in its advising strategy. The new approach encompasses a Life Mapping and Advising Model that builds faculty–student relationships and engages both parties effectively in the process. The model includes six important pillars to drive student success: (1) dedicated advising space, the Life Mapping and Advising Center (LMAC), (2) effective advisors, (3) integrated peer mentor and peer tutoring programs, (4) an intrusive advising strategy, (5) integration with first-year student success courses, and (6) life coaching. Although the program is in its infancy, based on the first-year assessment data, we have observed many promising trends that, together, point toward successful retention and persistence of our students in the major.

Acute myeloid leukemia (AML) often relapses following chemotherapy-induced remission and is generally chemo-resistant. Given the potential role for cancer stem cells in relapse, targeting of the leukemia-initiating cell (LIC) in AML may provide improved outcome following remission induction. However, due to overlap in their self-renewal program with normal hematopoietic stem cells (HSCs), therapeutic targeting of the LIC may have an adverse effect on long-term hematopoietic recovery. Here we used a mouse model of relapsed AML to explore whether the hypoxia-inducible factor (HIF)1α inhibitor echinomycin can be used to treat relapsed AML without affecting host HSCs. We show that echinomycin cured 40% to 60% of mice transplanted with relapsed AML. Bone marrow cells from the cured mice displayed normal composition of HSCs and their progenitors and were as competent as those isolated from nonleukemic mice in competitive repopulation assays. Importantly, in mice with complete remission, echinomycin appeared to completely eliminate LICs because no leukemia could be propagated in vivo following serial transplantation. Taken together, our data demonstrate that in a mouse model of relapsed AML, low-dose echinomycin selectively targets LICs and spares normal hematopoiesis.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and Flt3-ITD. MllPTD/WT:Flt3ITD/WT mice developed acute leukemia with 100% penetrance, at a median of 49 weeks. As in human MLL-PTD and/or the FLT3-ITD AML, mouse blasts exhibited normal cytogenetics, decreased Mll-WT-to-Mll-PTD ratio, loss of the Flt3-WT allele, and increased total Flt3. Highlighting the adverse impact of FLT3-ITD dosage on patient survival, mice with homozygous Flt3-ITD alleles, MllPTD/WT:Flt3ITD/ITD, demonstrated a nearly 30-week reduction in latency to overt AML. Here we demonstrate, for the first time, that Mll-PTD contributes to leukemogenesis as a gain-of-function mutation and describe a novel murine model closely recapitulating human AML.

One mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT-derived phenotypic short-term (ST)–HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

Linker for activation of T cells (LAT) plays a central role in T-cell activation by nucleating signaling complexes that are critical for the propagation of T-cell signals from the plasma membrane to the cellular interior. The role of phosphorylation and palmitoylation in LAT function has been well studied, but not much is known about other strategies by which the cell modulates LAT activity. We have focused on LAT ubiquitylation and have mapped the sites on which LAT is ubiquitylated. To elucidate the biological role of this process, we substituted LAT lysines with arginines. This resulted in a dramatic decrease in overall LAT ubiquitylation. Ubiquitylation-resistant mutants of LAT were internalized at rates comparable to wild-type LAT in a mechanism that required Cbl family proteins. However, these mutants displayed a defect in protein turnover rates. T-cell signaling was elevated in cells reconstituted with LAT mutants resistant to ubiquitylation, indicating that inhibition of LAT ubiquitylation enhances T-cell potency. These results support LAT ubiquitylation as a molecular checkpoint for attenuation of T-cell signaling.

In the last few years, great progress has been made in understanding how stromal interacting molecule 1 (STIM1), a protein containing a calcium sensor that is located in the endoplasmic reticulum, and Orai1, a protein that forms a calcium channel in the plasma membrane, interact and give rise to store-operated calcium entry. Pharmacological depletion of calcium stores leads to the formation of clusters containing STIM and Orai that appear to be sites for calcium influx. Similar puncta are also produced in response to physiological stimuli in immune cells. In T cells engaged with antigen-presenting cells, clusters containing STIM and Orai accumulate at the immunological synapse. We recently discovered that in activated T cells, STIM1 and Orai1 also accumulate in cap-like structures opposite the immune synapse at the distal pole of the cell. Both caps and puncta are long-lived stable structures containing STIM1 and Orai1 in close proximity. The function of puncta as sites of calcium influx is clear. We speculate that the caps may provide a secondary site of calcium entry. Alternatively, they may serve as a source of preformed channel complexes that move to new immune synapses as T cells repeatedly engage antigen-presenting cells.

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca2+influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca2+channel components to existing and newly forming immunological synapses.

Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together ∼16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

The morphogenesis of skin epithelia and adult hair follicle cycling both require integrated signaling between the epithelium and underlying mesenchyme. Because of their unique regulation, keratin intermediate filaments represent useful markers for the analysis of determination and differentiation processes in complex epithelia, such as the skin. In this study, we analyzed the distribution of mouse type I keratin 16 during skin morphogenesis, in the adult hair cycle, and in challenged epidermis. In mature hair follicles, we find keratin 16 along with its type II keratin partner keratin 6 in the companion layer of the outer root sheath during anagen and in the club hair sheath during catagen and telogen. During embryonic development, the distribution of keratin 16 is uncoupled from its presumed polymerization partner, keratin 6. Keratin 16 initially localizes within early hair germs, but rapidly shifts to a subset of cells at the interface of basal and suprabasal cells above and around the hair germ. The presence of keratin 16 at the transition between mitotically active and differentiating cells is recapitulated in primary keratinocytes cultured in vitro and in phorbol 12-myristate 13-acetate-treated back skin in vivo. We propose that keratin 16 marks cells in an intermediate state of cellular properties in which keratinocytes retain the flexibility required for activities such as cell migration and even mitosis but are resilient enough to provide the structural integrity required of the early suprabasal layers in the context of development, adult hair cycling, and wound repair.

Toward personalized therapy in AML: in vivo benefit of targeting aberrant epigenetics in MLL -PTD-associated AML

Keratins are the epithelial-specific members of the superfamily of intermediate filament (IF) genes and proteins. The 28 type I and 26 type II keratin genes are regulated in a pairwise fashion, reflecting the heteromeric nature of the 10 nm filaments they form, as well as in a tissue-specific and differentiation-dependent fashion in the body, giving rise to expression patterns that are very useful to define the status of epithelia in health and disease. Keratin IFs fulfill two major functions: structural support, without which incident physical trauma exposes an inherent fragility and leads to loss of cell integrity, and regulation of several basic cellular processes such as growth, proliferation, apoptosis, made possible by interactions with various cellular proteins.

Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS.

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 16 degrees C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

Publisher Summary Skin, by virtue of its location at the interface with the outside world, is one of the largest organs in the body that fulfills a number of important functions, undergoes constant renewal, and enjoys significant clinical relevance, as well as social importance. This chapter contains methods that are particularly useful for the basic characterization of transgenic mouse models in which the expression of keratin genes has been manipulated. Mouse skin presents several advantages for the study of the mechanisms regulating the expression of keratin genes and the properties, regulation, and function of their protein products, for example, a very large fraction of the known keratin genes are differentially expressed in skin epithelia and other IF genes are expressed in nonepithelial compartments of the skin. The compact nature of mouse keratin genes makes their manipulation through genetic engineering in relatively straightforward manner. The methods and assays outlined in the chapter are typically needed during the initial analysis of mice in which the expression of a gene, keratin, or other is manipulated in skin tissue. These methods, thus, have and will continue to be a mainstay in the characterization of the regulation and function of keratin genes in epithelial tissues.

The maize r locus encodes a transcription factor that regulates the developmental expression of the plant pigment anthocyanin. In an unusual example of gene regulatory diversity, the R-sc (Sc, strong seed color) and the R-p (P, plant color) alleles of r have nonoverlapping tissue specificity and nonhomologous 5′ flanking sequences. Heterozygotes between wild-type P and Sc mutants with Ds6 transposable element inserts (r-sc:m::Ds6 or sc:m) produce colored seed derivatives (Sc+) during meiotic recombination. The sc:m alleles with Ds6 insertion in 3′ regions of r produce crossover Sc+ derivatives. sc:m alleles with Ds6 elements inserted in 5′ regions produce rare Sc+ derivatives borne on nonrecombinant chromosomes. Among 52 such noncrossover Sc+ derivatives, 18 are indistinguishable from the Sc progenitor in phenotype and DNA sequence [Scp(+) alleles]. The remaining 34 derivatives have strong Sc+ expression, including darkly pigmented aleurone, scutellum, coleoptile, and scutellar node [Scp(e) alleles]. The coleoptile and scutellar node phenotypes are unique from either progenitor but are similar to those of some naturally occurring r alleles. Both classes of Sc+ derivatives are explained by gene conversion between the promoter region of Sc:124 and a homologous region located proximal to P. The recombinational intermediate formed between sc:m alleles and P results in deletion of the Ds6 element alone or both Ds6 and a nearby unrelated transposable element-like sequence.

The partial tandem duplication (PTD) of the MLL gene is a molecular defect in human myelodysplastic syndrome and acute myeloid leukemia (AML) and associates with poor patient outcomes, especially when other molecular prognostic markers are co-present such as the internal tandem duplication (ITD) of FLT3, i.e., FLT3-ITD. In human AML, MLL-PTD alters the epigenome via aberrant H3K4 methylation activity but is also associated with increased DNA methylation through an unknown mechanism (Whitman, et al, Blood 2008). Using a double knock-in mouse model of AML with Mll-PTD and Flt3-ITD, we analyzed the DNA methylome using a methyl binding protein (MBD2) pull down followed by next generation sequencing. Leukemic mouse bone marrow (BM, n=8) exhibited a 1.7 fold increase in global DNA methylation compared to non-leukemic controls (n=9, P=0.017). Consistent with this finding, DNA methyltransferases (Dmnt) 1, 3a, and 3b were overexpressed (1.5 fold P=0.03, 2.3 fold P=0.01, and 5.3 fold P=0.03, respectively). We previously showed that the non-coding microRNA 29b (miR-29b) expression directly downmodulates DNMT3a and 3b, but indirectly inhibits DNMT1 via miR-29b-driven SP1 suppression. In the murine Mll-PTD/Flt3-ITD AML, the precursor to the mature miR-29b (pri-miR-29b-2) was downregulated in leukemic BM (0.7-fold, P Citation Format: Kelsie M. Bernot, John S. Nemer, Ramasamy Santhanam, Shujun Liu, Gabriel G. Marcucci, Nicholas A. Zorko, Susan P. Whitman, Pearlly Yan, David Frankhouser, Ralf Bundschuh, Mengzi Zhang, Ronald F. Siebenaler, Elshafa H. Ahmed, Kathleen K. McConnell, Maura Munoz, Daniel L. Brook, Adrienne M. Dorrance, Katy E. Dickerson, Xiaoli Zhang, Jianying Zhang, David Jarjoura, Robert Lee, William Blum, Michael A. Caligiuri, Guido Marcucci. Targeting the miR29b/Sp1/Dnmt pathway for curative therapy in Mll-PTD/Flt3-ITD acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2013-3249

Cytogenetically normal acute myeloid leukemia (CN AML) patients with a MLL PTD have a poor prognosis compared to CN AML patients with MLL WT. We previously reported that the MLL WT gene in MLL PTD+ CN AML is epigenetically silenced. To investigate in vivo significance of the MLL PTD in the absence of MLL WT on hematopoiesis and leukemogenesis, we generated homozygous MllPTD/PTD and hemizygous MllPTD/- mice. These mice died in utero or as neonates, respectively, precluding further study on adult hematopioesis and leukemogenesis. In the current study, we crossed MllPTD/WT mice with Mll-conditional knock-out (cKO) animals to produce MllPTD/cKO mice. Like the MllPTD/WT mice, the MllPTD/cKO mice survive to adulthood, as both models express Mll WT and PTD. As previously reported by Jude et al, (Cell Stem Cell, 2007) Cre activation in hematopoietic cells of mice carrying the Mll cKO allele resulted in an intragenic deletion in Mll (deltaN allele) that when expressed, the protein was unable to translocate to the nucleus and thereby unable to exert normal Mll function. DNA PCR confirmed the correct MllPTD/deltaN genotype was generated. MlldeltaN/deltaN mice develop bone marrow (BM) failure (marked hypocellularity) at a median of 18 days post-Cre activation. Comparatively, MllPTD/deltaN mice (n=5) survived beyond 52 weeks post-Cre activation (P Citation Format: Nicholas A. Zorko, Daniel A. Yanes, W. Courtland Lewis, Daniel L. Brook, Kelsie M. Bernot, Ronald F. Siebenaler, Susan P. Whitman, Elshafa H. Ahmed, Kathleen K. McConnell, John Nemer, Patricia Ernst, Gang Huang, Guido Marcucci, Michael A. Caligiuri. The partial tandem duplication of Mll (Mll PTD) is a gain-of-function in the absence of Mll wildtype (Mll WT) in adult mouse hematopoiesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3849. doi:10.1158/1538-7445.AM2013-3849

Abstract 3501 Rearrangements of the Mixed-Lineage Leukemia (MLL) gene occur in a variety of aggressive human leukemias. The most common ones are balanced translocations in which the genomic sequences encoding the N-terminal portion of MLL are fused to sequences encoding the C-terminus of another translocation partner in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). Another mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and AML, but rarely seen in ALL, is partial tandem duplication (MLL-PTD). The MLL–PTD was first identified in de novo AML with a normal karyotype or trisomy 11. Cloning of this region revealed partial duplications within the 5′ region of the MLL gene. These duplications consist of an in-frame repetition of MLL exons in a 5′–3′ direction and produce an elongated protein. The incidence of MLL–PTD was 8% in unselected adult and childhood AML and 5% in MDS. However, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Mll-PTD knock-in mouse model, its expression is regulated by endogenous promoter, has been generated to study the function of Mll-PTD in vitro and in vivo and to identify its downstream targets. This mouse model provides a powerful genetic tool to identify disruptions in normal cellular regulation as a result of this mutation, as well as a model to characterize the contribution of the Mll-PTD in leukemogenesis. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state due to increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT–derived phenotypic short-term (ST)-HSCs/multipotent progenitors (MPPs) and granulocyte/macrophage progenitors (GMPs) have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. In conclusion, the MllPTD/WT mouse model provides unique genetic and biochemical tool to identify new targets and pathways responsible for the altered differentiation/repopulating properties, self-renewal activity, lineage bias and myeloid differentiation blockade relevant to MLL-PTD MDS and AML. This model should also help us to understand the underlying mechanism(s) for each of the phenotypes we found in this study and facilitate improved therapies and patient outcomes in the future. Disclosures: No relevant conflicts of interest to declare.

Abstract 2801 A relatively common mechanism through which Mixed-Lineage Leukemia (MLL) gene is disrupted in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) is a partial tandem duplication of its coding sequence (MLL-PTD), which duplicates the MLL N-terminal region and creates a larger in-frame oncogenic protein. We have previously shown that MLL interacts with and stabilizes the core binding factor (CBF) complex, a heterodimeric transcription factor complex composed of a DNA-binding subunit RUNX1 (CBFa) and a non-DNA-binding subunit CBFβ. MLL is required for CBF dependent regulation of the downstream target gene PU.11. In MDS and AML, RUNX1 is frequently disrupted by mutations, and the presence of MLL-PTD and/or RUNX1 mutations may negatively impact on clinical outcome. However, little is known how mutated MLL and RUNX1 contribute to MDS-associated ineffective hematopoiesis, clonal expansion and transformation into AML. Interestingly, MDS patients with either mutant RUNX1 or MLL-PTD tend to develop secondary AML harboring both RUNX1 and MLL mutations, suggesting a leukemogenic cooperation between the two genes through converging mechanisms. We hypothesized that the MLL-CBF nuclear signaling pathway maintains stem cell fitness, and the MDS-associated mutations co-opt this pathway leading to ineffective hematopoiesis, clonal expansion and transformation into AML. To address these questions, MLL-PTD+ and MLL-PTD(−) AML cell lines were compared. RUNX1 and CBFβ proteins were expressed at low levels in all 4 MLL-PTD+ AML cell lines (OCI-AML2, EOL, KOMP88, MUTZ11) compared with MLL-PTD(−) AML cell lines (OCI-AML5, Kasumi1, HL60, KG1a). In contrast to wild type MLL, which stabilized the CBF complex, MLL-PTD downregulated RUNX1 and CBFβ proteins in co-expression assays in 293T cells. This finding was further confirmed using the MllPTD/WT knock-in mouse model. In MllPTD/WT mice, the abnormal hematopoietic stem and progenitor cells (HSPCs) showed decreased expression levels of Runx1 (50%) and Cbfβ (30%) proteins and in turn PU.1 (30%) and its target gene, Mcl-1 (20%) compared with WT HSPCs. Consistent with this finding, HSPCs from MllPTD/WT mice had increased spontaneous apoptosis compared with WT HSPCs [(Lin−/cKit+/Sca-1+ (LSK), Annexin V positivity: 44% ±7.5% vs 25%±4.5%, p Disclosures: No relevant conflicts of interest to declare.