Loss Of Receptors Research Articles

5-LB: Rescuing Alpha-Cell Function in Type 1 Diabetes

Increased glucagon secretion from the pancreatic alpha cell is the first and most important defense against hypoglycemia. In T1D this defense mechanism is lost. We propose that the loss of the neighboring beta cells causes, and in turn the loss of inhibitory signals they generated, is a key factor in the inability of alpha cells to respond appropriately to blood sugar levels. The sudden cessation of this inhibitory input during hypoglycemia is a necessary signal for the glucagon response. We hypothesize that reactivation of endogenous paracrine and autocrine signaling might restore the alpha cell’s ability to respond to hypoglycemia in T1D. We used isolated islets and tissue slices from non-diabetic organ donors and donors with T1D and measured dynamic hormone secretion and calcium recordings. We found that alpha cell responses are briefer than the sustained beta cell responses. If not reset by inhibitory input, the alpha cell is not able to respond again. Glucagon secretion can be recovered by activation of paracrine signals to inhibit the alpha cell. We found full glucagon recovery if reset for 15 min or longer using high glucose in healthy islets (no reset 0.51 +/- 0.09 vs. 5 min reset 0.99 +/- 0.12 vs. 15 min 1.63 +/- 0.31, fold change to baseline +/- SEM). In tissues from T1D donors, alpha cells failed to respond to decreases in glucose concentration despite normal glucagon content and responses to KCl depolarization. Baseline glucagon secretion was significantly elevated compared to healthy individuals (mean 25.93 +/- SEM 8.55 pM in T1D vs 7.49 +/- 2.21, p<0.05). Furthermore, we found severely diminished Ca2+ responses to both lowering glucose concentration and glutamate receptor stimulation. We demonstrate that alpha cells from T1D donors cannot mount an efficient glucagon response due to deficient glutamate receptor signaling and loss of paracrine inhibitory input. Our results suggest that restoring both signals rescues glucagon secretion. Disclosure J. Panzer: None. A. Pugliese: Consultant; Provention Bio, Inc. Funding American Diabetes Association (4-22-PDFPM-12 to J.P.); The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053, G-2108-04793)

Basal type I interferon signaling has only modest effects on neonatal and juvenile hematopoiesis.

Type I interferon (IFN-1) regulates gene expression and hematopoiesis both during development and in response to inflammatory stress. We previously showed that during development in mice, hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) induce IFN-1 target genes shortly before birth. This coincides with the onset of a transition to adult hematopoiesis, and it drives the expression of genes associated with antigen presentation. However, it is not clear whether perinatal IFN-1 modulates hematopoietic output, as has been observed in contexts of inflammation. We have characterized hematopoiesis at several different stages of blood formation, from HSCs to mature blood cells, and found that loss of the IFN-1 receptor (IFNAR1) leads to depletion of several phenotypic HSC and MPP subpopulations in neonatal and juvenile mice. Committed lymphoid and myeloid progenitor populations expand simultaneously. These changes had a surprisingly little effect on the production of more differentiated blood cells. Cellular indexing of transcriptomes and epitopes by sequencing resolved the discrepancy between the extensive changes in progenitor numbers and modest changes in hematopoiesis, revealing stability in most MPP populations in Ifnar1-deficient neonates when the populations were identified based on gene expression rather than surface marker phenotype. Thus, basal IFN-1 signaling has only modest effects on hematopoiesis. Discordance between transcriptionally and phenotypically defined MPP populations may affect interpretations of how IFN-1 shapes hematopoiesis in other contexts, such as aging or inflammation.

Abstract B057: ZBTB7A as a novel vulnerability in neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a lethal variant of aggressive prostate cancer. Hallmarks of NEPC include the loss of androgen receptor (AR) signaling and transdifferentiation toward small-cell neuroendocrine (SCN) phenotypes, which result in resistance to AR-targeted therapy. NEPC resembles other SCN carcinomas clinically, histologically and genomically. Here, we leveraged SCN phenotype scores of various cancer cell lines and gene depletion screens from the Cancer Dependency Map (DepMap) to identify vulnerabilities in NEPC. We discovered ZBTB7A, a transcription factor, as a candidate promoting the progression of NEPC. Cancer cells with high SCN phenotype scores showed a strong dependency with RET kinase, an emerging therapeutic target of NEPC, suggesting the NEPC-like phenotype of the cells. Then, we observed a strong correlation between dependencies of RET and ZBTB7A in these cells. Utilizing informatic modeling of whole transcriptome sequencing data from patient samples, we revealed distinct gene networking patterns of ZBTB7A in NEPC versus prostate adenocarcinoma. Specifically, we observed a robust association of ZBTB7A with genes promoting cell cycle progression in NEPC, and this result was confirmed through Gene Set Enrichment Analysis. Furthermore, we showed a potential interaction of ZBTB7A with apoptosis regulating genes. Silencing ZBTB7A in a NEPC cell line confirmed the dependency of cells on ZBTB7A for cell growth, and the role of ZBTB7A in regulating G1/S transition in cell cycle and inducing apoptosis. Collectively, our results highlight the oncogenic function of ZBTB7A in NEPC, and emphasize the value of ZBTB7A as a promising therapeutic strategy for targeting NEPC tumors. Citation Format: Song Yi Bae, Hannah E. Bergom, Abderrahman Day, Joseph T. Greene, Tanya S. Freedman, Justin H. Hwang, Justin M. Drake. ZBTB7A as a novel vulnerability in neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B057.

Loss of toll-like receptor 5 potentiates spontaneous hepatocarcinogenesis in farnesoid X receptor-deficient mice.

HCC is the most common primary liver cancer and a leading cause of cancer-related mortality. Gut microbiota is a large collection of microbes, predominately bacteria, that harbor the gastrointestinal tract. Changes in gut microbiota that deviate from the native composition, that is, "dysbiosis," is proposed as a probable diagnostic biomarker and a risk factor for HCC. However, whether gut microbiota dysbiosis is a cause or a consequence of HCC is unknown. To better understand the role of gut microbiota in HCC, mice deficient of toll-like receptor 5 (TLR5, a receptor for bacterial flagellin) as a model of spontaneous gut microbiota dysbiosis were crossed with farnesoid X receptor knockout mice (FxrKO), a genetic model for spontaneous HCC. Male FxrKO/Tlr5KO double knockout (DKO), FxrKO, Tlr5KO, and wild-type (WT) mice were aged to the 16-month HCC time point. Compared with FxrKO mice, DKO mice had more severe hepatooncogenesis at the gross, histological, and transcript levels and this was associated with pronounced cholestatic liver injury. The bile acid dysmetabolism in FxrKO mice became more aberrant in the absence of TLR5 due in part to suppression of bile acid secretion and enhanced cholestasis. Out of the 14 enriched taxon signatures seen in the DKO gut microbiota, 50% were dominated by the Proteobacteria phylum with expansion of the gut pathobiont γ-Proteobacteria that is implicated in HCC. Collectively, introducing gut microbiota dysbiosis by TLR5 deletion exacerbated hepatocarcinogenesis in the FxrKO mouse model.

Incongruence between transcriptional and vascular pathophysiological cell states

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

Carfentanil is a β-arrestin-biased agonist at the μ opioid receptor.

The illicit use of fentanyl-like drugs (fentanyls), which are μ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls. For agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Agonist-induced cell surface receptor loss was assessed using an enzyme-linked immunosorbent assay, whilst agonist-induced G protein-coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the μ opioid receptor were determined in silico using molecular dynamics simulations. Relative to the reference ligand DAMGO, carfentanil was β-arrestin-biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein-coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias. Carfentanil is a β-arrestin-biased opioid drug at the μ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: A case for diminished GDNF-signaling

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), which represents a time point of initiating GDNF treatment later than reported in some preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemiparkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) hemilesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred, returning to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors thus appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance statementAlthough preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, there is uncertainty if it can alleviate motor impairment in Parkinson's disease patients. Using the established 6-OHDA hemiparkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery.

Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure.

Patients on mechanical circulatory support are prone to infections, increasing morbidity and mortality. These circulatory support devices generate high mechanical shear stress (HMSS) that can causes trauma to blood. When leukocytes become damaged, their immune response function may be impaired or weakened, leading to increased infection vulnerability. This study examined neutrophil structural and functional alterations after exposure to 75, 125, and 175 Pa HMSS for 1 second. Human blood was exposed to three levels of HMSS using a blood shearing device. Neutrophil morphological alteration was characterized by examining blood smears. Flow cytometry assays were used to analyze expression levels of CD62L and CD162 receptors, activation level (CD11b), and aggregation (platelet-neutrophil aggregates). Neutrophil phagocytosis and rolling were examined via functional assays. The results show neutrophil structure (morphology and surface receptors) and function (activation, aggregation, phagocytosis, rolling) were significantly altered after HMSS exposure. These alterations include cell membrane damage, loss of surface receptors (CD62L and CD162), initiation of activation and aggregation, upregulation of phagocytic ability and increased rolling speed. The alterations were the most severe after 175 Pa exposure. HMSS caused damage and activation of neutrophils, potentially impairing normal neutrophil function, leading to weakened immune defense and increasing a patient's vulnerability to infections.

Insulin signaling is critical for sinoatrial node maintenance and function

Insulin and insulin-like growth factor 1 (IGF-1) signaling regulate cellular growth and glucose metabolism in the myocardium. However, their physiological role in the cells of the cardiac conduction system has never been explored. Therefore, we sought to determine the spatiotemporal function of insulin/IGF-1 receptors in the sinoatrial node (SAN). We generated cardiac conduction cell-specific inducible IGF-1 receptor (IGF-1R) knockout (KO) (CSIGF1RKO), insulin receptor (IR) KO (CSIRKO), and IR/IGF-1R double-KO (CSDIRKO) mice and evaluated their phenotypes. Telemetric electrocardiography revealed regular sinus rhythm in CSIGF1RKO mice, indicating that IGF-1R is dispensable for normal pacemaking. In contrast, CSIRKO and CSDIRKO mice exhibited profound sinus bradycardia. CSDIRKO mice showed typical sinus node dysfunction characterized by junctional rhythm and sinus pauses on electrocardiography. Interestingly, the lack of an insulin receptor in the SAN cells of CSIRKO and CSDIRKO mice caused sinus nodal fibrosis. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) protein expression significantly decreased in the CSIRKO and CSDIRKO mice relative to the controls. A patch-clamp study of the SAN cells of CSIRKO mice revealed a significant decrease in the funny current, which is responsible for spontaneous diastolic depolarization in the SAN. This result suggested that insulin receptor loss reduces the heart rate via downregulation of the HCN4 channel. Additionally, HCN1 expression was decreased in CSDIRKO mice, explaining their sinus node dysfunction. Our results reveal a previously unrecognized role of insulin/IGF-1 signaling in sinus node structural maintenance and pacemaker function.

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition.

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.

Anaplastic Kaposi Sarcoma: A Clinicopathologic and Molecular Genetic Analysis

Kaposi sarcoma (KS) is a human herpesvirus 8 (HHV8)-associated vascular proliferation that most often involves the skin. Rarely, KS shows marked nuclear atypia or pleomorphism; such examples are known as “anaplastic” KS. This poorly characterized variant often pursues an aggressive course; little is known of its genetic landscape. This study evaluated the clinicopathologic and genomic features of anaplastic KS. We identified 9 anaplastic KS cases from 7 patients and 8 conventional KS cases, including a matched conventional KS and primary metastasis anaplastic KS pair from a single patient (anaplastic KS diagnosed 9 years after conventional KS). All patients with anaplastic KS were men, aged 51 to 82 years, who had locally aggressive tumors predominantly affecting the soft tissue and bone of the lower extremities (5/7 patients). Four patients were known to be HIV positive (all on antiretrovirals), 2 were HIV negative, and 1 was of unknown HIV status. The tumors showed angiosarcoma-like or pleomorphic spindle cell sarcoma morphology. Plasma cell–rich chronic inflammation and hemosiderin deposition were commonly present. Single-nucleotide polymorphism–based chromosomal microarray analysis showed the anaplastic KS cohort to demonstrate highly recurrent whole chromosome (chr) gains of chr 7, 11, 19, and 21, which primarily affected olfactory and G protein–coupled receptor signaling and losses of chr6_q and chrY. Compared with conventional KS, anaplastic KS cases showed significantly more total copy number alterations and more frequent gains of chr7 and chr11_q13.1 (MARK2, RELA, and ESRRA, including high copy number gain in 1 case). Pathway analysis demonstrated that these gains preferentially affected genes that facilitate cyclin-dependent cell signaling. Furthermore, anaplastic KS cases were phylogenetically distinct from conventional KS cases, including the patient-matched primary metastasis anaplastic KS pair and conventional KS. Our study is the first to demonstrate that a more complex genome and distinct copy number alterations distinguish anaplastic KS from conventional KS. Gains of chr7 and chr11_q13.1 appear central to biological transformation.

Evidence for a protective effect of the loss of α4-containing nicotinic acetylcholine receptors on Aβ-related neuropathology in Tg2576 mice.

Loss of cholinergic neurons as well as α4β2* (* = containing) nicotinic acetylcholine receptors (nAChRs) is a prominent feature of Alzheimer's disease (AD). Specifically, amyloid β (Aβ), the principal pathogenic factor of AD, is a high affinity ligand for nAChRs. Yet, the pathophysiological role of nAChRs in AD is not well established. In the present study, we have investigated the effects of the loss of α4* nAChRs on the histological alterations of the Tg2576 mouse model of AD (APPswe) crossing hemizygous APPswe mice with mice carrying the genetic inactivation of α4 nAChR subunit (α4KO). A global decrease in Aβ plaque load was observed in the forebrain of APPswe/α4KO mice in comparison with APPswe mice, that was particularly marked in neocortex of 15 month-old mice. At the same age, several alterations in synaptophysin immunoreactivity were observed in cortico-hippocampal regions of APPswe mice that were partially counteracted by α4KO. The analysis of the immunoreactivity of specific astroglia (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adapter molecule, Iba1) markers showed an increase in the number as well as in the area occupied by these cells in APPswe mice that were partially counteracted by α4KO. Overall, the present histological study points to a detrimental role of α4* nAChRs that may be specific for Aβ-related neuropathology.

Microglia actively remove NR1 autoantibody-bound NMDA receptors and associated post-synaptic proteins in neuron microglia co-cultures.

Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood. Here, using a patient-derived monoclonal NR1 autoantibody (hNR1-mAb) and a co-culture system of microglia and neurons, we could show that hNR1-mAb bound to hippocampal neurons led to microglia-mediated removal of hNR1-mAb bound NMDARs. These complexes were found to accumulate inside endo-lysosomal compartments of microglia. Utilizing another patient isolated monoclonal autoantibody, against the α1-subunit of GABAA receptors (α1-GABAA -mAb), such removal of receptors was found to be specific to the antibody-bound receptor targets. Interestingly, along with receptor removal, we also observed a reduction in synapse number, more specifically in the numbers of post-synaptic proteins like PSD95 and Homer 1, when microglia were present in the culture. Importantly, mutations in the Fc region of hNR1-mAb, blocking its Fcγ receptor (FcγR) and complement binding, attenuated hNR1-mAb driven loss of NMDARs and synapses, indicating that microglia engagement by bound hNR1-mAb is critical for receptor and synapse loss. Our data argues for an active involvement of microglia in removal of NMDARs and other receptors in individuals with autoimmune encephalitis, thereby contributing to the etiology of these diseases.

Abstract 5315: Identification of urinary proteome signatures associated with triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, accounts for ~15% of all breast cancers and is associated with a poor prognosis and high rates of recurrence and distant metastases. Patients diagnosed with TNBC have limited treatment options and are often restricted to chemotherapy-based regimens, in stark contrast to patients with other breast cancer subtypes where the development of targeted therapeutics have significantly improved patient outcomes. TNBC is characterized by the loss of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. This and the genetic and molecular tumor heterogeneity of TNBC has not only limited advances in the development of targeted therapy but also in the identification of biomarkers that can predict disease status and monitor treatment response. Advances in next-generation sequencing technologies have led to the identification of promising clinically actionable mutations however these tests often require invasive procedures to obtain tumor tissue and lack the ability to monitor treatment efficacy or detect recurrence, highlighting the need for non-invasive markers. A number of serum protein biomarkers have been evaluated in TNBC however none have yet to be successfully deployed in the clinic as indicators of disease. We have previously established that the urinary proteome can serve as a source of highly specific and sensitive biomarkers that can detect disease status and stage of multiple cancer types. Within the context of our global proteomics approach to cancer biomarker discovery, we have utilized the SomaScan discovery platform to identify 281 proteins that are differentially expressed in the urine of patients with TNBC compared to the urine of age- and sex-matched controls. The gene expression profiles of these proteins in TNBC tumor tissue were compared to those of normal breast tissue utilizing the R2 Genomics Platform. Proteins whose gene expression profile was consistent with the proteomic data were validated for the ability to distinguish between TNBC and non-TNBC diagnoses using mono-specific ELISAs of patient urine samples. These results demonstrate that a global proteomics approach coupled with clinical gene expression data can be effectively utilized to identify potential biomarkers for TNBC. These findings identify clinically relevant proteins that should be further explored as potential biomarkers in the development of non-invasive diagnostics for TNBC. (This work was supported by the Breast Cancer Research Foundation and the Nile Albright Research Foundation). Citation Format: Michael N. Lombardo, Roopali Roy, Susan Pories, Meg Lotz, Rama Aldakhlallah, Simon T. Dillon, Towia A. Libermann, Marsha A. Moses. Identification of urinary proteome signatures associated with triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5315.

Evolutionary loss of the β1-adrenergic receptor in salmonids

Whole-genome duplications (WGDs) have been at the heart of the diversification of β-adrenergic receptors (β-ARs) in vertebrates. Non-teleost jawed vertebrates typically possess three β-AR genes: adrb1 (β1-AR), adrb2 (β2-AR), and adrb3 (β3-AR), originating from the ancient 2R (two rounds) WGDs. Teleost fishes, owing to the teleost-specific WGD, have five ancestral adrb paralogs (adrb1, adrb2a, adrb2b, adrb3a and adrb3b). Salmonids are particularly intriguing from an evolutionary perspective as they experienced an additional WGD after separating from other teleosts. Moreover, adrenergic regulation in salmonids, especially rainbow trout, has been intensively studied for decades. However, the repertoire of adrb genes in salmonids has not been yet characterized. An exhaustive genome survey of diverse salmonids, spanning five genera, complemented by phylogenetic sequence analysis, revealed each species has seven adrb paralogs: two adrb2a, two adrb2b, two adrb3a and one adrb3b. Surprisingly, salmonids emerge as the first known jawed vertebrate lineage to lack adrb1. adrb1 is nevertheless highly expressed in the hearts of non-salmonid teleosts, indicating that the wealth of data on adrenergic regulation in salmonids should be generalised to other teleost fishes with caution. It is hypothesised that the loss of adrb1 could have been viable because of the evolutionary radiation of adrb2 and adrb3 genes attributable to the salmonid WGD.

ZBTB7A as a novel vulnerability in neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer. NEPC is characterized by the loss of androgen receptor (AR) signaling and transdifferentiation toward small-cell neuroendocrine (SCN) phenotypes, which results in resistance to AR-targeted therapy. NEPC resembles other SCN carcinomas clinically, histologically and in gene expression. Here, we leveraged SCN phenotype scores of various cancer cell lines and gene depletion screens from the Cancer Dependency Map (DepMap) to identify vulnerabilities in NEPC. We discovered ZBTB7A, a transcription factor, as a candidate promoting the progression of NEPC. Cancer cells with high SCN phenotype scores showed a strong dependency on RET kinase activity with a high correlation between RET and ZBTB7A dependencies in these cells. Utilizing informatic modeling of whole transcriptome sequencing data from patient samples, we identified distinct gene networking patterns of ZBTB7A in NEPC versus prostate adenocarcinoma. Specifically, we observed a robust association of ZBTB7A with genes promoting cell cycle progression, including apoptosis regulating genes. Silencing ZBTB7A in a NEPC cell line confirmed the dependency on ZBTB7A for cell growth via suppression of the G1/S transition in the cell cycle and induction of apoptosis. Collectively, our results highlight the oncogenic function of ZBTB7A in NEPC and emphasize the value of ZBTB7A as a promising therapeutic strategy for targeting NEPC tumors.

Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution.MethodWe generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T.ResultsOur nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T.ConclusionCD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.

Evolution of biological features of invasive lobular breast cancer: Comparison between primary tumour and metastases

BackgroundInvasive lobular carcinoma (ILC) has unique clinical-biological features. Phenotypical differences between primary tumours (PTs) and metastases (M) have been described for invasive ductal carcinoma, but data on ILC are limited. MethodsWe retrospectively analysed patients with recurrent ILC from our institution from 2013 to 2020. We evaluated the discordance of the oestrogen receptor (ER), progesterone receptor (PgR) and HER2 between PT and M, to understand prognostic and therapeutic implications. ResultsThirteen percent (n = 91) of all patients had ILC. We observed 15%, 44% and 5% of ER, PgR and HER2 status discordance between PT and M. ER/PgR discordance was related to receptor loss and HER2 mainly due to gain. PT presented a luminal-like phenotype (93%); 6% and 1% were triple-negative (TNBC) and HER2-positive. In M, there was an increase in TNBC (16%) and HER2-positive (5%). Metastasis-free survival and overall survival (OS) were different according to clinical phenotype, with poorer prognosis for HER2+ and TNBC (p < 0.001); OS after metastatic progression did not differ across phenotypes (p = 0.079). In luminal-like ILC (n = 85) at diagnosis, we found that OS after relapse was poorer in patients experiencing a phenotype switch to TNBC but improved in patients with HER2 gain (p = 0.0028). Poorer survival was reported in patients with a PgR and/or ER expression loss of ≥25%. There was HER2-low enrichment in M1 (from 37% to 58%): this change was not associated with OS (p > 0.05). ConclusionOur results suggest that phenotype switch after metastatic progression may be associated with patients’ outcomes. Tumour biopsy in recurrent ILC could drive treatment decision-making, with prognostic implications.

Abstract P6-11-07: Role of insulin resistance in HR+ mammary carcinogenesis

Abstract Hormone receptor (HR+) breast cancer (BC) is responsible for more than 80% BC cases and more than 60% BC-related deaths in the US. The incidence and severity of BC are influenced by a variety of modifiable risk factors, including (but not limited to) nutritional behaviors and obesity. Specifically, women with a body mass index (BMI) &amp;gt; 25 Kg/m2 exhibit an approx. 1.4-fold increase in the risk of developing postmenopausal HR+ BC as compared to women with BMI &amp;lt; 25 Kg/m2. Along similar lines, women with type 2 diabetes (T2D) – one of many consequences of obesity – are at 1.23-fold increased risk to develop postmenopausal HR+ BC as compared to women without T2D. Moreover, a BMI &amp;gt;30 Kg/m2 has been associated with decreased progression-free survival and overall survival (OS) in women with HR+ BC. Thus, a high number of postmenopausal HR+ BC cases and BC-related deaths could be effectively avoided by modifying dietary habits, both in prophylactic and therapeutic settings. Mechanistically, obesity provokes numerous alterations that have been linked to accrued postmenopausal HR+ carcinogenesis, encompassing (1) increased circulating levels of glucose and insulin – reflecting insulin resistance (IR) coupled to T2D, (2) increased levels of estrogen, produced locally and systematically by adipocytes, and (3) chronic inflammation of the breast adipose tissue (AT). However, the relative contribution of IR vs. other obesity-associated alterations to BC development and sensitivity to treatment has not been elucidated. At least in part, this reflects the two most common approaches employed to cause obesity in preclinical studies: the use of high-fat diets (HFDs) and/or mice genetically predisposed to develop obesity (e.g., ob/ob mice), neither of which is suitable to uncouple IR from all other metabolic, hormonal, and inflammatory effects of obesity. Here, we combined a unique model of HR+HER2- carcinogenesis that recapitulates key immunobiological features of human HR+ BC (notably, a cold tumor microenvironment coupled to a scarce sensitivity to immunotherapy, and exquisite sensitivity to CDK4/6 inhibitors), as established in mice by medroxyprogesterone acetate (M) pellets combined with 7,12-dimethylbenz[a]anthracene (D) oral administration, with a novel model of pure IR originating from the AT-specific deletion of RAB10, member RAS oncogene family (Rab10), a transducer of insulin signaling in adipocytes. Importantly, these mice are neither hyperphagic nor overweight, have normal glucose level at baseline and do not exhibit inflammatory alterations in the AT, but nonetheless display IR. While a HFD shortens tumor-free survival (TFS) in immunocompetent mice subjected to M/D-driven oncogenesis, it does not alter the growth of detectable M/D-driven tumors. Conversely, IR as imposed by the loss of Rab10 in the AT failed to alter TFS but accelerated the growth of tumors, resulting in shortened OS. Moreover, the deletion of Rab10 from the mouse AT enabled the development of tumors displaying features of increased aggressiveness, including systematic loss of progesterone receptor (PR) expression. Finally, the growth of tumors emerging in in the context of a Rab10/- AT could be efficiently controlled by the systemic administration of metformin. In line with this notion, orthotopically injected mouse triple negative BC AT-3 cells grew faster in mice bearing an AT-specific deletion of Rab10 as compared to their wild-type littermates. In conclusion, IR as imposed by the loss of Rab10 in the mouse AT converts HR+ mammary carcinomas as driven by M/D into HR- lesions with increased aggressiveness. These findings have major implications for ethnic groups that are at high risk for T2D at BMIs that are considered normal for Caucasians, such as women of African American and Asian descent, especially in view of the fact that women from these ethnic groups are known to be at increased risk for aggressive triple-negative BC as compared to their Caucasian counterparts. Citation Format: Aitziber Buque, Maria Belen Picatoste, Norma Bloy, Lucie Yammine, Rosemary Leahey, Ai Sato, Emma Johnson, Timothy E. McGraw, Lorenzo Galluzzi. Role of insulin resistance in HR+ mammary carcinogenesis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-07.

Abstract P3-05-08: Prevalence and prognosis of ER-loss in advanced invasive lobular carcinoma

Abstract Introduction: Estrogen receptor (ER) loss occurs in about 20% of recurrent breast cancers (BC) and is associated with unresponsiveness to endocrine therapy (ET) and poor prognosis. Prior studies evaluating ER-loss included predominately patients with invasive ductal carcinoma (IDC), and therefore the impact of ER-loss in invasive lobular carcinoma (ILC) is unknown. In this retrospective analysis, using real-world data, we aimed to determine the prevalence and clinical significance of ER-loss in ILC. Methods: Advanced BC were molecularly profiled at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing of DNA (592-gene panel or whole-exome sequencing), RNA (whole transcriptome sequencing, WTS) and immunohistochemistry (IHC) of select markers. A large real-world evidence (RWE) database combining Caris’ molecular data with clinical information obtained from insurance claims data (CODEai) was interrogated and overall survival (OS) was calculated from time of tissue collection to last patient contact. A tumor was considered to have ER-loss if therapies approved only for ER-positive BC were prescribed prior to obtaining a negative ER IHC result. OS was compared using Kaplan-Meier estimates for defined patient cohorts; significance was determined as p values &amp;lt; 0.05. For molecular analyses, Fisher-Exact or Chi-Square tests were used to determine p values. Correction for multiple comparisons was performed using Benjamini-Hochberg to calculate q values. Results: The RWE database included 24,824 patients with advanced BC. At the time of tissue collection for molecular profiling, 6,786 advanced BC patients had been previously treated with ET (with or without mTOR or CDK4/6 inhibitors), of whom 1,338 had data available on histologic classification and ER IHC. The final analytical cohort included 263 patients with ILC and 1,075 with IDC. ER-loss was identified in 11.4% of ILC (n=30/263) and 19.6% (n=210/1075) of IDC (p=0.0017). In ILC, ER-loss was associated with significantly worse OS (HR: 1.75, 95%CI: 1.10-2.79, p=0.016) compared with no ER-loss. In the cohort of patients with ER-loss, patients with ILC had significantly worse OS compared with IDC (HR=2.03, 95% CI: 1.267-3.251, p=0.003). Further, when 1,016 tumors with ER-loss (regardless of histology) were stratified by the median OS (mOS=11mo), positive PD-L1 expression (34% vs. 22%, p=0.04, q=0.22), HER2 IHC positivity (16% vs. 7.8%, p=0.003, q=0.08) and HER2 amplification (16% vs. 4.7%, p=0.0006, q=0.04) were enriched in patients with longer mOS; while amplification of TEFB (0.38% vs. 2.6%, p=0.047, q=0.23) and MYB (0.38% vs. 2.6%, p=0.047, q=0.23) were enriched in patients with shorter mOS. WTS identified 197 differentially expressed genes, the majority of which were enriched in patients with longer mOS (q&amp;lt; 0.05). Conclusions: In this large real-word dataset, ER-loss likely occurred in 11.4% of ILC and was associated with worse OS compared to both patients with IDC and ER-loss and ILC without ER-loss. Our analysis had several limitations; notably, our definition of ER-loss was based on prior treatment, we could not distinguish between de novo or recurrent metastatic disease and time of tissue collection was not standardized during the course of treatment. Thus, additional studies are needed to confirm these findings. However, this study does suggest that ER-loss occurs in a subset of patients with ILC and has poor prognostic implications. Citation Format: Whitney L. Hensing, Joanne Xiu, W. Michael Korn, Stephanie L. Graff, Irene Kang, Evanthia T. Roussos Torres, Arielle L. Heeke, Andrew A. Davis, Nusayba A. Bagegni, Katherine K. Clifton, Ron Bose, Cynthia Ma, Foluso O. Ademuyiwa. Prevalence and prognosis of ER-loss in advanced invasive lobular carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-08.