A novel peptidomimetic therapeutic for selective suppression of lung cancer stem cells over non-stem cancer cells.

Cancers are highly heterogeneous and typically contain a small subset of drug-resisting cells called tumor initiating cells or cancer stem cells (CSCs). CSCs can self-renew, divide asymmetrically, and often cause tumor invasion and metastasis. Therefore, treatments specifically targeting CSCs are critical to improve patient survival. Recently, we identified a highly specific peptidomimetic (peptoid - PCS2) that selectively binds to the CSC subpopulation of lung cancer over the remaining cancer cells (non-CSCs). Subsequently, we identified plectin as the target of PCS2. Plectin is an intracellular structural protein, which is involved in tumor invasion and metastasis when it appears on cell surface. While PCS2 monomer did not display any anti-cancer activity, we designed a series of homo-dimeric versions of PCS2, and identified PCS2D1.2 optimized homo-dimer that displayed highly specific cytotoxicity towards CSCs over non-CSCs. PCS2D1.2 effectively blocked the in vitro colony formation and cell migration, hallmarks of CSCs. Furthermore, PCS2D1.2 reduced the in vivo tumor formation. In both in vitro and in vivo studies, PCS2D1.2 effectively reduced plectin expression and/or plectin-rich CSCs, but had no effect on non-CSCs. Therefore, PCS2D1.2 has the potential to be developed as a highly CSC specific drug candidate, which can be used in combination with current anti-cancer drugs.

Unbiased peptoid combinatorial cell screen identifies plectin protein as a potential biomarker for lung cancer stem cells.

Tumors often contain a small subset of drug-resisting, self-renewing, and highly metastatic cells called tumor initiating cells or cancer stem cells (CSCs). To develop new approaches to detecting and targeting lung cancer CSCs, we applied an "unbiased" peptoid combinatorial cell screen to identify highly specific ligands that bind a CSC subpopulation of non-small cell lung cancer cells (defined by Aldefluor positivity), but not the remaining aldefluor negative cancer cells from the same preclinical model. One of the 'hit' peptoids bound to plectin, a structural protein, predominantly expressed intracellularly, but whose localization on the cell surface is linked to tumor invasion and metastasis. Our studies show both genotypic and phenotypic correlations between plectin and lung CSCs, as well as association of high plectin mRNA expression with poor patient survival in lung adenocarcinoma, potentially identifying plectin as a biomarker for lung CSCs.

A role for BMP-induced homeobox gene MIXL1 in acute myelogenous leukemia and identification of type I BMP receptor as a potential target for therapy.

Mesoderm Inducer in Xenopus Like1 (MIXL1), a paired-type homeobox transcription factor induced by TGF-ß family of ligands is required for early embryonic specification of mesoderm and endoderm. Retrovirally transduced Mixl1 is reported to induce acute myelogenous leukemia (AML) with a high penetrance. But the mechanistic underpinnings of MIXL1 mediated leukemogenesis are unknown. Here, we establish the protooncogene c-REL to be a transcriptional target of MIXL1 by genome wide chromatin immune precipitation. Accordingly, expression of c-REL and its downstream targets BCL2L1 and BCL2A2 are elevated in MIXL1 expressing cells. Notably, MIXL1 regulates c-REL through a zinc finger binding motif, potentially by a MIXL1-Zinc finger protein transcriptional complex. Furthermore, MIXL1 expression is detected in the cancer genome atlas (TCGA) AML samples in a pattern mutually exclusive from that of HOXA9, CDX2 and HLX suggesting the existence of a core, yet distinct HOX transcriptional program. Finally, we demonstrate MIXL1 to be induced by BMP4 and not TGF-ß in primary human hematopoietic stem and progenitor cells. Consequently, MIXL1 expressing AML cells are preferentially sensitive to the BMPR1 kinase inhibitor LDN-193189. These findings support the existence of a novel MIXL1-c REL mediated survival axis in AML that can be targeted by BMPR1 inhibitors. (MIXL1- human gene, Mixl1- mouse ortholog, MIXL1- protein).

Requirement for ssbp2 in hematopoietic stem cell maintenance and stress response.

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.

Cardiac response to startle stimuli in larval zebrafish: sympathetic and parasympathetic components.

Central regulation of cardiac output via the sympathetic and parasympathetic branches of the autonomic nervous system allows the organism to respond to environmental changes. Sudden onset stimuli, startle stimuli, are useful probes to study central regulatory responses to the environment. In mammals, startle stimuli induce a transient bradycardia that habituates with repeated stimulation. Repeated presentation of the stimulus results in tachycardia. In this study, we investigate the behavioral regulation of heart rate in response to sudden stimuli in the zebrafish. Larval zebrafish show a stereotyped heart rate response to mild electrical shock. Naïve fish show a significant increase in interbeat interval that resolves in the 2 s following stimulation. This transient bradycardia decreases on repeated exposure to the stimulus. Following repeated stimulation, the fish become tachycardic within 1 min of stimulation. Both the transient bradycardia and following tachycardia responses are blocked with administration of the ganglionic blocker hexamethonium, demonstrating that these responses are mediated centrally. The transient bradycardia is blocked by the muscarinic antagonist atropine, suggesting that this response is mediated by the parasympathetic system, while the following tachycardia is specifically blocked by the beta-adrenergic antagonist propranolol, suggesting that this response is mediated by the sympathetic nervous system. Together, these results demonstrate that at the larval stage, zebrafish actively regulate cardiac output to changes in their environment using both the parasympathetic and sympathetic branches of the autonomic nervous system, a behavioral response that is markedly similar to that observed in mammals to similar sudden onset stimuli.