John A. Latham

Cross-regulation of histone modifications

Histones undergo several different post-translational modifications that control a variety of physiological processes. These covalent modifications show substantial cross-regulation, providing a wealth of regulatory potential. New insights into the communication between modifications on histones have emerged in recent years. This review assesses the current understanding of cross-regulation of histone modifications and identifies future questions to be addressed in this field.

The Set1 Methyltransferase Opposes Ipl1 Aurora Kinase Functions in Chromosome Segregation

A balance in the activities of the Ipl Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. We report here that this balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. We find that Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. Our studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, our findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function.

Chromatin Signaling to Kinetochores: Transregulation of Dam1 Methylation by Histone H2B Ubiquitination

Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory crosstalk in trans between a histone modification and modification on a nonhistone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries.

Induction of Migraine-Like Photophobic Behavior in Mice by Both Peripheral and Central CGRP Mechanisms.

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. Although migraine can be treated using CGRP antagonists that act peripherally, the relevant sites of CGRP action remain unknown. To address the role of CGRP both within and outside the CNS, we used CGRP-induced light-aversive behavior in mice as a measure of migraine-associated photophobia. Peripheral (intraperitoneal) injection of CGRP resulted in light-aversive behavior in wild-type CD1 mice similar to aversion seen previously after central (intracerebroventricular) injection. The phenotype was also observed in C57BL/6J mice, although to a lesser degree and with more variability. After intraperitoneal CGRP, motility was decreased in the dark only, similar to motility changes after intracerebroventricular CGRP. In addition, as with intracerebroventricular CGRP, there was no general increase in anxiety as measured in an open-field assay after intraperitoneal CGRP. Importantly, two clinically effective migraine drugs, the 5-HT1B/D agonist sumatriptan and a CGRP-blocking monoclonal antibody, attenuated the peripheral CGRP-induced light aversion and motility behaviors. To begin to address the mechanism of peripheral CGRP action, we used transgenic CGRP-sensitized mice that have elevated levels of the CGRP receptor hRAMP1 subunit in nervous tissue (nestin/hRAMP1). Surprisingly, sensitivity to low light was not seen after intraperitoneal CGRP injection, but was seen after intracerebroventricular CGRP injection. These results suggest that CGRP can act in both the periphery and the brain by distinct mechanisms and that CGRP actions may be transmitted to the CNS via indirect sensitization of peripheral nerves. SIGNIFICANCE STATEMENT The neuropeptide calcitonin gene-related peptide (CGRP) is a central player in migraine pathogenesis, yet its site(s) of action remains unknown. Some preclinical studies have pointed to central sites in the brain and brainstem. However, a peripheral site of action is indicated by the ability of intravenous CGRP to trigger migraine in humans and the efficacy of CGRP receptor antagonists that evidently do no penetrate the CNS in effective amounts. Resolving this issue is particularly important given recent clinical trials showing that anti-CGRP monoclonal antibodies can reduce and even prevent migraine attacks. In this study, we report that CGRP can act in both the brain and the periphery of the mouse to cause migraine-like photophobia by apparently distinct mechanisms.

ALD1613, a Novel Long-Acting Monoclonal Antibody to Control ACTH-Driven Pharmacology

Adrenocorticotropic hormone (ACTH) is the primary regulator of adrenal glucocorticoid production. Elevated levels of ACTH play a critical role in disease progression in several indications, including congenital adrenal hyperplasia and Cushing disease. We have generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1613) to ACTH. In vitro, ALD1613 neutralizes ACTH-induced signaling via all 5 melanocortin receptors and inhibited ACTH-induced cyclic adenosine monophosphate accumulation in a mouse adrenal cell line (Y1). ALD1613 administration to wild-type rats significantly reduced plasma corticosterone levels in a dose-dependent manner. In rodent models with either chronic infusion of ACTH or acute restraint stress-induced ACTH, corticosterone levels were significantly reduced by ALD1613. Administration of ALD1613 to nonhuman primates on days 1 and 7 stably reduced plasma cortisol levels >50% for 57 days. ALD1613 demonstrates the potential of a monoclonal antibody to be an effective therapeutic for conditions with elevated ACTH levels.

Abstract 1217: Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cachexia is present in the majority of patients with advanced cancer progression and is not reversible through nutritional supplementation, leading to loss of skeletal muscle and adipose tissue. There are a lack of quality pre-clinical models for cachexia research. Previously, Champions Oncology reported a mouse cachexia model derived from a primary human renal cell carcinoma tumor implanted in immunocompromised mice. This model, CTG-0804, was developed using the innovative Champions TumorGraft™ platform, which preserves the biological properties of the original human tumor. The Champions TumorGraft platform demonstrated dramatic weight loss in these mice as the tumor grew. Plasma from these mice also showed elevated levels of human IL-6. We have now further characterized this TumorGraft model by evaluating standard of care (SOC) agents for renal cell carcinoma and anti-IL-6 compounds to determine the mechanism of cachexia. The SOC evaluation revealed that sorafenib, sunitinib, temsirolimus and bevacizumab as single agents provided a survival advantage by inhibiting tumor growth and preventing weight loss in these mice. Blockade of human IL-6 with tocilizumab or ALD518 did not inhibit tumor growth and weight loss still occurred, demonstrating the possibility of other pathways involved in the cachexia mechanism. In addition, the SOC data for sunitinib showed significant tumor growth inhibition, but no regression at the study end point. This correlated with the clinical profile, where sunitinib could not prevent metastasis and the patient eventually progressed. The TumorGraft model was molecularly characterized and mutation analysis revealed a KIT V530I mutation. This mutation has been reported in extra-abdominal aggressive fibromatosis (desmoid tumor) and acute myeloid leukemia patients that have responded to c-kit targeting therapies, such as imatanib and dasatinib. Further characterization of this TumorGraft is ongoing to include additional bioinformatics using gene expression and mutation pathway analyses. In summary, we have continued development of this cachexia model using the Champions TumorGraft platform, along with molecular evaluation, to produce a robust and well-characterized approach for cachexia investigation which can also be applied to other areas of drug development and cancer research. Citation Format: Nathan Anderson, Tin Khor, Andrew Feldhaus, Andreya Gatling, Katie Olson, John Latham, David Sidransky, Elizabeth M. Bruckheimer. Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1217. doi:10.1158/1538-7445.AM2014-1217

Abstract 2792: Characterization of spontaneous in vivo cachexia models in Champions TumorGraft™ models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cachexia, defined as a loss of adipose tissue and skeletal muscle mass not reversible by nutritional support, is present in the majority of patients with advanced cancer progression. This reduces the patients quality of life and impacts their ability to function normally and undergo treatments. Reversing cachexia has been problematic due to a lack of quality pre-clinical models. Traditional xenograft models using cell lines are limited due to their inability to accurately replicate the whole body response to cancer. Champions Oncology overcomes this challenge through the use of its innovative Champions TumorGraft™ platform, where primary human tumors are implanted into immunocompromised mice in a manner that preserves the biological properties of the original human tumor. Champions has identified a panel of three TumorGraft models which spontaneously induce cachexia when implanted subcutaneously into nude mice: 1) CTG-0282, a pancreatic cancer model, 2) CTG-0765, a NSCLC model, and 3) CTG-0804, a renal cell carcinoma model. All three models have shown a greater than 90% cachexia rate, as demonstrated by lost body weight, over multiple passages. In addition, loss of body weight correlates with an increase in tumor volume size. Mice that have little or no tumor growth show no loss of body weight. Past studies have indicated a function for pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6) and interferon gamma (IFNγ), in the cachexia process. Champions is currently investigating the roles of these possible tumor-mediated factors in the aforementioned models. Principle among these factors is IL-6, which has been shown to be required in the regulation of cachexia in tumor-bearing mice and is correlated with weight loss and survival in patients. Preliminary results from our studies suggest a correlation between IL-6 levels and spontaneous cachexia in Champions TumorGraft models. In summary, we demonstrate that the Champions TumorGraft platform preserves the biological properties of the original human tumor, including spontaneous cachexia behavior in these models, and is therefore ideal for oncology drug development programs focused on the inhibition of cachexia. Citation Format: Nathan Anderson, Tin Oo Khor, Andrew Feldhaus, Katie Olson, John Latham, David Sidransky, Elizabeth M. Bruckheimer. Characterization of spontaneous in vivo cachexia models in Champions TumorGraft™ models. [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 2792. doi:10.1158/1538-7445.AM2013-2792

Abstract IA07: New functions for histone modifying enzymes

Histone modifying enzymes are important creating and maintaining epigenetic programs that regulate cell identity and growth. Mutations or abnormal expression of these proteins are associated with multiple cancers. Our lab uses genetic approaches to define the full spectrum of functions for these enzymes. For example, we have created a series of mutations in the mouse Gcn5 gene in order to define the functions of this histone acetyltransferase (HAT) in a mammalian system. Deletion of Gcn5 led to early embryonic death and to telomere dysfunction. Biochemical studies revealed that depletion of Gcn5 or ubiquitin specific protease 22 (Usp22), which is another bona fide component of the Gcn5-containing SAGA-type complex, increases the turnover of two shelterin proteins, TRF1 and POT1a (Atanassov, 2009). Our studies provided the first indication that Gcn5 and mammalian SAGA influence telomere maintenance and the first demonstration that SAGA affects protein stability. We are now defining the role of Gcn5 and SAGA in maintenance of pluripotency in ES cells and ES cell differentiation. Our data indicate that Gcn5 is an important cofactor for both Myc and E2F family transcription factors. We are also identifying additional USP22 protein substrates and are defining USP22 functions during mouse development. We have also discovered unexpected functions for the Set1 lysine methyltransferase, which is highly homologous with the MLL protein, during mitosis. We found that Set1 methylates kinetochore proteins, such as Dam1, in yeast, and we have determined that H2Bub is required in trans for methylation of this protein. This work provides the first example of cross talk between a histone modification and a modification in a non-histone protein, defining a new mode of “chromatin signaling.” Zhang K, Lin W, Latham JA, Riefler GM, Schumacher JM, Chan C, Tatchell K, Hawke DH, Kobayashi R, Dent SY. The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation. Cell 122(5):723-34, 9/2005. PMCID: PMC1794220 Atanassov BS, Evrard YA, Multani AS, Zhang Z, Tora L, Devys D, Chang S, Dent SY. Gcn5 and SAGA Regulate Shelterin Protein Turnover and Telomere Maintenance. Mol Cell 35(3):352-364, 8/2009. PMCID: PMC2749492. Atanassov BS, Dent SY. USP22 regulates cell proliferation by deubiquitinating the transcriptional regulator FBP1. EMBO Rep 12(9):924-30, 9/2011. e-Pub 9/2011. PMCID: PMC3166460. Latham JA, Chosed RJ, Wang S, Dent SY. Chromatin signaling to kinetochores: Trans-regulation of Dam1 methylation by histone H2B ubiquitination. Cell 146(5):709-19, 9/2011. PMCID: PMC3168986. Citation Format: Sharon Y.R. Dent, Boyko Atanassov, Calley Hirsch, Evangelia Koutelou, John Latham. New functions for histone modifying enzymes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA07.

The H2BK123Rgument: Table I.

The discovery of trans-regulation of histone H3K4 methylation by ubiquitination of histone H2BK123 generated much excitement in the field of chromatin biology. Recently, the veracity of this example of cross talk between histone modifications in yeast was challenged (Foster and Downs, 2009. J. Cell Biol. doi:10.1083/jcb.200812088) but ultimately reconfirmed in a study in this issue (Nakanishi et al., 2009. J. Cell Biol. doi:10.1083/jcb.200906005).