Nelson-Holte, Molly

Abstract Background X and Y chromosome analysis is a critical component of genetic testing, used both diagnostically and as a quality control (QC) metric. Discordances between expected and observed sex chromosome data can arise due to mislabeling, demographic data errors, transplant history, or biological variations. Such discordances pose challenges to laboratories and affect patient care, particularly in marginalized populations and unique clinical contexts. Methods We reviewed cases of sex chromosome discordance identified at our laboratory from January 2021 through August 2023. Cases spanned various testing methods and were categorized by the root cause, including mislabeling, sample mix-ups, transgender individuals, stem cell transplants, and unexplained causes. Case outcomes were assessed, and potential resolutions were analyzed. Results Among 65 cases identified, the leading cause of discordance was mislabeling (n = 20, 31%), followed by other/not identified (n = 16, 25%), sample mix-ups (n = 13, 20%), transgender individuals (n = 9, 14%), and stem cell transplants (n = 7, 11%). Cases required additional QC processes such as reanalysis, clinician contact, and occasionally sample re-collection. The process extended turnaround times by up to 13 business days. Detailed case reviews highlighted the challenges and implications of managing these discordances, emphasizing the importance of accurate data transmission and inclusive practices. Conclusion Using X and Y chromosome data as a QC metric can identify critical errors but also introduces limitations and bias. Improved standardization, inclusive practices, and alternative QC methods are necessary to ensure accuracy and equitable patient care. Collaborative efforts are required to address demographic complexities and reduce testing delays.

Abstract Background: Epigenetic based therapies, such as histone deacetylase inhibitors, have proven successful in many cancer types including lymphomas and myelomas with ongoing phase III clinical trials in NSCLC. Epigenetic modulation in triple negative breast cancer (TNBC) has been increasingly explored in the past decade. A major issue preventing the uptake of these drugs in the clinic is the relatively high doses necessary to achieve therapeutic benefit. It is therefore of interest to identify combinatorial approaches that result in drug de-escalation and enhanced therapeutic benefit. We and others have demonstrated that estrogen receptor beta (ERβ) is expressed in approximately 20% of TNBC patients. ERβ targeted therapies, including estradiol and multiple ERβ selective agonists, result in suppression of cell proliferation, invasion, and migration in vitro and in vivo (Reese, PNAS 2018). For these reasons, we sought to determine if ERβ targeted therapies would synergize with epigenetic based therapies for the treatment of TNBC. Methods: Using multiple TNBC cell lines representative of ERβ+ and ERβ- disease, as well as a model of acquired resistance to ERβ targeted therapies, we performed an epigenetic drug screen assessing 145 compounds alone and in combination with estradiol treatment. The top hit from this screen was extensively profiled across multiple cell lines in the presence and absence of ERβ targeting agents and was compared to the efficacy observed in ERα+ models of breast cancer using cell proliferation assays, RT-PCR, and western blotting. Results: The most significant compound to elicit differential and therapeutically beneficial effects in ERβ+ TNBC was CUDC-101, a multitarget pan-HDACi, EGFRi, and HER2i that was developed based on the structures of Vorinostat, Erlotinib, and Lapatinib. CUDC-101 potently inhibits the growth of TNBC cells and synergizes with E2 stimulation in ERβ+ MDA-MB-231 TNBC cells, but not in ERβ- cells. CUDC-101 was also effective in ERβ-resistant MDA-MB-231 cells and restored the growth inhibitory effects of estrogen in this model. Treatment with Vorinostat (SAHA) alone elicited a similar, though far less potent, synergistic effect in the MDA-MB-231-ERβ+ cell line, suggesting that histone deacetylase inhibition is a critical, but not completely sufficient, component of the synergistic phenotype. CUDC-101, while effective at reducing the viability of MDA-MB-468-ERβ+ cell line, had no synergistic effect with E2 in this model suggesting differences in response between TNBC sub-types. Additionally, CUDC-101 was found to be ineffective in models of ERα+ disease (MCF7 and MCF7-ERβ) in both the presence and absence of E2 treatment. Conclusions: Therapeutic options remain extremely limited for TNBC patients and the development of novel treatment strategies for these patients continue to represent a major unmet clinical need. Here, we present evidence that CUDC-101 elicits potent anti-cancer effects in multiple models of TNBC and synergizes with ERβ-targeted therapies in some models. Our findings further strengthen the body of evidence suggesting ERβ as a valid therapeutic target and important biomarker in TNBC and further implicate HDACi/EGFRi as relevant therapeutic strategies within this patient population. Citation Format: Michael Justin Emch, David Benjamin, Elizabeth Bruinsma, Molly Nelson-Holte, Matthew Goetz, John Hawse. CUDC-101 enhances estrogen-mediated anticancer effects in ERβ+ TNBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-10.

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.

Abstract Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.