Medical School Without Walls: 50 Years of Regional Campuses at Indiana University School of Medicine.

The history of Indiana University School of Medicine (IUSM) dates to 1871, when Indiana Medical College entered into an affiliation with Indiana University in Bloomington to offer medical education. In 1971, the Indiana General Assembly passed a bill to create and fund a distributed model for medical education for which IUSM was responsible, an innovative approach to implementing a statewide medical education program. IUSM became one of the first U.S. medical schools to implement what is today known as a regional medical campus model. This regional medical campus system has permitted IUSM to expand enrollment based on national and local concerns about physician shortages, increase access to care locally, support expansion of graduate medical education, and provide opportunities for research and scholarship by faculty and students statewide. This effort was made possible by partnerships with other universities and health care systems across the state and the support of local community and state leaders. The model is a forward-thinking and cost-effective way to educate physicians for service in the state of Indiana and is applicable to others. This article highlights milestones in IUSM's 50-year history of regional medical education, describes the development of the regional medical campus model, recognizes significant achievements over the years, shares lessons learned, and discusses considerations for the future of medical education.

HoxA9 binds and represses the Cebpa +8 kb enhancer.

C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav-NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav-NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis.

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

The protective role of DOT1L in UV-induced melanomagenesis.

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.

A new target for differentiation therapy in AML.

Despite major advances in understanding the genetics and epigenetics of acute myelogenous leukemia, there is still a great need to develop more specific and effective therapies. High throughput approaches involving either genetic approaches or small molecule inhibitor screens are beginning to identify promising new therapeutic targets.

Deregulation of the HOXA9/MEIS1 axis in acute leukemia.

PURPOSE OF REVIEW: HOXA9 is a homeodomain transcription factor that plays an essential role in normal hematopoiesis and acute leukemia, in which its overexpression is strongly correlated with poor prognosis. The present review highlights recent advances in the understanding of genetic alterations leading to deregulation of HOXA9 and the downstream mechanisms of HOXA9-mediated transformation. RECENT FINDINGS: A variety of genetic alterations including MLL translocations, NUP98-fusions, NPM1 mutations, CDX deregulation, and MOZ-fusions lead to high-level HOXA9 expression in acute leukemias. The mechanisms resulting in HOXA9 overexpression are beginning to be defined and represent attractive therapeutic targets. Small molecules targeting MLL-fusion protein complex members, such as DOT1L and menin, have shown promising results in animal models, and a DOT1L inhibitor is currently being tested in clinical trials. Essential HOXA9 cofactors and collaborators are also being identified, including transcription factors PU.1 and C/EBPα, which are required for HOXA9-driven leukemia. HOXA9 targets including IGF1, CDX4, INK4A/INK4B/ARF, mir-21, and mir-196b and many others provide another avenue for potential drug development. SUMMARY: HOXA9 deregulation underlies a large subset of aggressive acute leukemias. Understanding the mechanisms regulating the expression and activity of HOXA9, along with its critical downstream targets, shows promise for the development of more selective and effective leukemia therapies.

PRDM16 Suppresses MLL1r Leukemia via Intrinsic Histone Methyltransferase Activity.

PRDM16 is a transcription co-factor that plays critical roles in development of brown adipose tissue, as well as maintenance of adult hematopoietic and neural stem cells. Here we report that PRDM16 is a histone H3K4 methyltransferase on chromatin. Mutation in the N-terminal PR domain of PRDM16 abolishes the intrinsic enzymatic activity of PRDM16. We show that the methyltransferase activity of PRDM16 is required for specific suppression of MLL fusion protein-induced leukemogenesis both in vitro and in vivo. Mechanistic studies show that PRDM16 directly activates the SNAG family transcription factor Gfi1b, which in turn downregulates the HOXA gene cluster. Knockdown Gfi1b represses PRDM16-mediated tumor suppression, while Gfi1b overexpression mimics PRDM16 overexpression. In further support of the tumor suppressor function of PRDM16, silencing PRDM16 by DNA methylation is concomitant with MLL-AF9-induced leukemic transformation. Taken together, our study reveals a previously uncharacterized function of PRDM16 that depends on its PR domain activity.

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo.

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

The same site on the integrase-binding domain of lens epithelium-derived growth factor is a therapeutic target for MLL leukemia and HIV.

Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.

Targeting recruitment of disruptor of telomeric silencing 1-like (DOT1L): characterizing the interactions between DOT1L and mixed lineage leukemia (MLL) fusion proteins.

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.

MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction.

MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target.

MLL-ENL inhibits polycomb repressive complex 1 to achieve efficient transformation of hematopoietic cells.

Stimulation of transcriptional elongation is a key activity of leukemogenic MLL fusion proteins. Here, we provide evidence that MLL-ENL also inhibits Polycomb-mediated silencing as a prerequisite for efficient transformation. Biochemical studies identified ENL as a scaffold that contacted the elongation machinery as well as the Polycomb repressive complex 1 (PRC1) component CBX8. These interactions were mutually exclusive in vitro, corresponding to an antagonistic behavior of MLL-ENL and CBX8 in vivo. CBX8 inhibited elongation in a specific reporter assay, and this effect was neutralized by direct association with ENL. Correspondingly, CBX8-binding-defective MLL-ENL could not fully activate gene loci necessary for transformation. Finally, we demonstrate dimerization of MLL-ENL as a neomorphic activity that may augment Polycomb inhibition and transformation.

A subset of mixed lineage leukemia proteins has plant homeodomain (PHD)-mediated E3 ligase activity.

The mixed lineage leukemia protein MLL1 contains four highly conserved plant homeodomain (PHD) fingers, which are invariably deleted in oncogenic MLL1 fusion proteins in human leukemia. Here we show that the second PHD finger (PHD2) of MLL1 is an E3 ubiquitin ligase in the presence of the E2-conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4, the closest homolog to MLL1 but not in MLL2 or MLL3. Mutation of PHD2 leads to MLL1 stabilization, as well as increased transactivation ability and MLL1 recruitment to the target gene loci, suggesting that PHD2 negatively regulates MLL1 activity.

Twenty-first century pathology sign-out.

It is difficult to imagine a field that is changing as rapidly as pathology. A convergence of factors including not only scientific and technological advances but also changes in business models is transforming the field, particularly in the area of cancer diagnostics. The authors examine 8 themes, or "forces of change," in pathology and speculate on how these will affect pathology sign-out and the future role of pathologists in patient care.

Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia.

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.