LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

Two adjacent C/EBP-binding sequences that participate in the cell-specific expression of the mouse serum amyloid A3 gene.

Serum amyloid A (SAA) is a major acute-phase protein synthesized primarily in the liver. Its expression, very low in normal animals, is increased several hundredfold following acute inflammation. To examine DNA sequences involved in liver-specific expression, 5'-flanking regions of the mouse SAA3 gene were analyzed by transient transfection, band shift, and DNase I protection assays. We found that a 56-bp fragment immediately 5' to the TATA box spanning the region -93 to -38 relative to the transcription start site was sufficient to confer liver cell-specific transcriptional activation onto a heterologous promoter in a dose-dependent and orientation-independent manner. This DNA fragment could form DNA-protein complexes with heat-stable nuclear proteins, and the complexes formed could be specifically competed for by excess oligomers corresponding to the C/EBP- or DBP-binding sites but not by binding sites for three other liver-specific factors, HNF1, HNF3, and HNF4. Footprint analysis using Hep3B nuclear extracts revealed two adjacent footprint regions within this 56-bp fragment, the distal region having at least fivefold-greater affinity than the proximal region. Identical footprint patterns were observed when purified recombinant C/EBP protein was used. These results indicated that binding of C/EBP to this 56-bp fragment plays an important role in vivo in enhancing expression of the mouse SAA3 gene in hepatocytes.

Regulation of mouse serum amyloid A gene expression in transfected hepatoma cells.

Expression of mouse serum amyloid A (SAA1, -2, and -3) mRNAs can be induced up to 1,000-fold in the liver in response to acute inflammation. This large increase is primarily the result of a 200-fold increase in the rates of SAA gene transcription. To analyze the cis-acting regulatory element(s) responsible for regulating transcription, we fused 306 base pairs of the mouse SAA3 promoter to a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and transfected this chimeric DNA into cultured cells. In transient expression assays, this 5' sequence was sufficient to confer cell-specific expression: CAT activity was readily detectable when the construct was transfected into liver-derived cells but was not detectable in nonliver cells. Furthermore, when liver cells transfected with this construct were treated with conditioned media prepared from activated mixed lymphocyte cultures or with recombinant interleukin-1, a 10- to 15-fold increase in CAT activity was detected. Deletion analyses showed two regions of interest: a proximal region that enhanced CAT expression in a cell-specific manner and a distal region that conferred responsiveness to both conditioned media and recombinant interleukin-1. This distal responsive element had properties of an inducible transcriptional enhancer, and deletion of the proximal cell-specific region rendered the distal element responsive to stimulation by conditioned media in nonliver cells.

Molecular and cellular biology of serum amyloid A.

Serum amyloid A (SAA) is one of the major acute-phase proteins in humans and mice. It is synthesized predominantly by the liver and secreted as a major component of the apolipoproteins in the high density lipoprotein particle. While the major physiological function of SAA is unclear, prolonged elevation of plasma SAA levels, as in chronic inflammation, however, results in the pathological condition amyloidosis affecting the liver, kidney and spleen. The expression of SAA mRNA is dramatically elevated in response to infection or systemic inflammation and is due primarily to the increased rate of SAA gene transcription. Studies in vitro and in vivo demonstrated that the expression of SAA genes is regulated by the inflammatory cytokine interleukin-1. Moreover, both the interleukin-1-induced expression and the enhanced liver-specific expression of the SAA gene are controlled by the binding of nuclear proteins to specific DNA sequences upstream from the structural gene.

Identification of a transcriptional enhancer in a mouse amyloid gene.

We have employed a mouse liver-derived cell line (BNL) to identify DNA sequences in the promoter of the serum amyloid A3 (SAA3) gene essential for expression. In both transient DNA transfection assays and BNL cells stably transformed with SAA3 fusion genes, deletion analysis of the SAA3 promoter indicates that sequences between -185 and -138 base pairs 5' to the transcription initiation site are essential for expression of heterologous genes. A 69-base pair sequence spanning this region markedly augmented expression of the bacterial chloramphenicol transacetylase gene regardless of orientation or position. The homology of this sequence with sequences in the promotors of other genes expressed by liver during inflammation suggests a common mechanism of regulation.

Regulation of amyloid A gene expression in cultured cells.

Serum amyloid A (SAA) proteins are secreted by mammalian liver in response to inflammatory stimuli. Both transcriptional and posttranscriptional mechanisms have been shown to regulate the 2,000-fold increase in SAA mRNA after injection of endotoxin into mice. We report here the characterization of a cell line derived from mouse liver (BNL) in which the expression of SAA3 mRNA is regulated. In this model, SAA3 mRNA accumulated in response to conditioned medium from the mouse macrophage P388D1 cell line with kinetics similar to that seen in mouse liver (C. A. Lowell, R. S. Stearman, and J. F. Morrow, J. Biol. Chem. 261:8453-8461, 1986). In in vitro nuclear transcription assays, the SAA3 gene was transcribed equally in induced and uninduced cells. In addition, in steady-state RNA studies treatment with conditioned medium allowed the cells to rapidly accumulate SAA3 mRNA without an apparent change in half-life. These observations suggest that conditioned medium contains a factor(s) that acts directly on hepatocytes to regulate SAA3 mRNA processing.

Effect of deep surgical sepsis on protein-sparing therapies and nitrogen balance.

Four patients from a larger group of 18 patients receiving dextrose-free isotonic (3%) amino acid solution as nutritional support, form the basis of this report. An additional seven patients received intravenous isotonic (5%) dextrose as their sole support in the postoperative period following major elective surgery (average nitrogen balance = -12.3 +/- 2.7 g). All patients were well-nourished as determined by anthropometric measurements. The nonseptic patients receiving infusions of isotonic amino acids demonstrated an improvement in nitrogen balance (= delta 8.5 +2, P less than 0.001) when compared to the postoperative use of 100 to 150 g of glucose. However, sepsis produced a decreased net utilization of the infused crystalline amino acids such that nitrogen balance was similar to the intravenous glucose group (- 10.6 +/- 2.1). This septic response was associated with decreased plasma free fatty acid concentrations and the absence of starvation ketosis and ketonuria. While the nitrogen balance was not different in the septic and the dextrose control groups, deficiencies in plasma amino acid concentrations were observed in the group receiving intravenous infusion of glucose.