Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.cancer | aCGH | A-myb

Alterations of BRAF are the most common known genetic aberrations in pediatric gliomas. They frequently are found in pilocytic astrocytomas, where genomic duplications involving BRAF and the poorly characterized gene KIAA1549 create fusion proteins with constitutive B-Raf kinase activity. BRAF V600E point mutations are less common and generally occur in nonpilocytic tumors. The development of BRAF inhibitors as drugs has created an urgent need for robust clinical assays to identify activating lesions in BRAF. KIAA1549-BRAF fusion transcripts have been detected in frozen tissue, however, methods for FFPE tissue have not been reported. We developed a panel of FFPE-compatible quantitative RT-PCR assays for the most common KIAA1549-BRAF fusion transcripts. Application of these assays to a collection of 51 low-grade pediatric gliomas showed 97% sensitivity and 91% specificity compared with fluorescence in situ hybridization or array comparative genomic hybridization. In parallel, we assayed samples for the presence of the BRAF V600E mutation by PCR pyrosequencing. The data further support previous observations that these two alterations of the BRAF, KIAA1549 fusions and V600E point mutations, are associated primarily with pilocytic astrocytomas and nonpilocytic gliomas, respectively. These results show that fusion transcripts and mutations can be detected reliably in standard FFPE specimens and may be useful for incorporation into future studies of pediatric gliomas in basic science or clinical trials.

Stat5a is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that directly target Stat5a, despite ample evidence that it contributes to breast cancer pathogenesis. Here, deacetylation of the Stat5a coactivator and chromatin remodeling protein HMGN2 on lysine residue K2 by HDAC6 promotes Stat5a-mediated transcription and breast cancer growth. HDAC6 inhibition both in vitro and in vivo enhances HMGN2 acetylation with a concomitant reduction in Stat5a-mediated signaling, resulting in an inhibition of breast cancer growth. Furthermore, HMGN2 is highly acetylated at K2 in normal human breast tissue, but is deacetylated in primary breast tumors and lymph node metastases, suggesting that targeting HMGN2 deacetylation is a viable treatment for breast cancer. Together, these results reveal a novel mechanism by which HDAC6 activity promotes the transcription of Stat5a target genes and demonstrate utility of HDAC6 inhibition for breast cancer therapy. Implications HMGN2 deacetylation enhances Stat5a transcriptional activity, thereby regulating prolactin-induced gene transcription and breast cancer growth.

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

Summary Prolactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Context. In advanced cancer, abnormal sleep patterns may contribute to poor quality of life, but the impact of opioidrelated sleep disorders has not been explored in detail in these patients. Objective. To document sleep and respiratory patterns in patients with cancer, receiving a range of opioids, determine factors that contribute to severity of central or obstructive apnea, and to what extent these contribute to sleep disturbance. Methods. Adults with advanced cancer admitted to a palliative care service underwent a sleep analysis by an unattended polysomnography. Total sleep time, apnea hypopnea index, central apnea index, obstructive apnea hypopnea index, arousal index, and oxygen desaturation were measured. Baseline assessment included body habitus, Mallampati score, comorbidity indices, concomitant medications, and the Berlin questionnaire. Epworth Sleepiness Scale, Stanford Sleepiness Scale, and Wu cancer fatigue scales were documented. Results. Twenty-eight patients were studied, including 25 receiving opioids. In the latter group, the apnea hypopnea index was mildly abnormal in six patients and severely abnormal in 10 patients. Central apnea index and obstructive apnea hypopnea index were abnormal in nine and 17 patients, respectively. There was no significant correlation between opioid dose and polysomnographic results. Conclusion. In patients with advanced cancer receiving opioid analgesia, there was a high prevalence of respiratory disturbance, both central and obstructive, and deranged sleep patterns. Addressing sleep-disordered breathing in cancer patients has the potential to improve daytime drowsiness and quality of life.

The hormone, prolactin, has been implicated in breast cancer pathogenesis and regulates chromatin engagement by the transcription factor, STAT5A. STAT5A is known to inducibly bind promoters and cis-regulatory elements genome wide, though the mechanisms by which it exerts specificity and regulation of target gene expression remain enigmatic. We previously identified HDAC6 and HMGN2 as cofactors that facilitate prolactin induced, STAT5A mediated gene expression. Here, multi-condition STAT5A, HDAC6, and HMGN2 ChIP-seq with parallel condition RNA-seq are utilized to reveal the cis-regulatory landscape and cofactor dynamics underlying prolactin stimulated gene expression in breast cancer. We find that prolactin regulated genes are significantly enriched for cis-regulatory elements bound by HDAC6 and HMGN2, and that inducible STAT5A binding at enhancers, rather than promoters, conveys specificity for prolactin regulated genes. The selective HDAC6 inhibitor, ACY-241, blocks prolactin induced STAT5A chromatin engagement at cis-regulatory elements as well as a significant proportion of prolactin stimulated gene expression. We identify functional pathways known to contribute to the development and/or progression of breast cancer that are activated by prolactin and inhibited by ACY-241. Additionally, we find that the DNA sequences underlying shared STAT5A and HDAC6 binding-sites at enhancers are differentially enriched for estrogen response elements (ESR1 and ESR2 motifs) relative to enhancers bound by STAT5A alone. Gene set enrichment analysis identifies significant overlap of ERα regulated genes with genes regulated by prolactin, particularly prolactin regulated genes with promoters or enhancers co-occupied by both STAT5A and HDAC6. Lastly, the therapeutic efficacy of ACY-241 is demonstrated in in vitro and in vivo breast cancer models, where we identify synergistic ACY-241 drug combinations and observe differential sensitivity of ER + models relative to ER - models.

Stat5 is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that target Stat5, despite ample evidence that it contributes to breast cancer pathogenesis. Previous research in our lab has shown that the high mobility group nucleosome binding domain 2 (HGMN2) protein serves as a Stat5 co-activator. The activity of HMGN2 has been previously shown to be regulated by acetylation. Here, we show that deacetylation of HMGN2 on lysine residue K2 by histone deacetylase 6 (HDAC6) promotes Stat5-mediated transcription and breast cancer growth. Conversely, in vitro HDAC6 inhibition by pharmacologic and knockdown approaches enhanced HMGN2 acetylation with a concomitant reduction of in vitro Stat5-mediated signaling and global gene expression, and breast cancer growth. These data, combined with global in silico ChIP-Seq analysis, indicate that HDAC6 serves as a regulator of the pioneer function of HMGN2 for Stat5 transcriptional function. In vitro and in vivo treatment of traditional and patient-derived xenograft models with HDAC6 inhibitors resulted in a highly significant reduction of tumor growth. Translationally, it was also found that high levels of acetylated K2 were present in normal human breast tissue, which was lost in primary breast cancers and lymph node metastases. This suggests that blockade of HMGN2 deacetylation as described above is a novel treatment for breast cancer, given that existing HDAC6 inhibitors have a favorable toxicity profile in phase I trials of other tumor types. Altogether, these results reveal a novel mechanism through which HDAC6 regulates the transcription of Stat5 target genes and demonstrates the utility of HDAC6 inhibition as a potential breast cancer therapeutic. Citation Format: Justin M. Craig, Suzanne M. Schauwecker, Charles V. Clevenger. HDAC6 as a therapeutic target in human breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B11.