Biology subjects

Perkins, B.

Publications and source records attributed to Perkins, B..

10 recordsLinked to original sources

bioRxiv2025

A PBD-dimer containing antibody drug conjugate targeting CCRL2 for high-risk MDS/AML

Patients with myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) with high-risk features including TP53 mutations and deletions have poor outcomes due to lack of effective therapies. The atypical chemokine surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in MDS and secondary AML (sAML) compared to healthy hematopoietic cells and we recently found that TP53-mutated MDS/AML and AML with erythroid features express the highest levels of this receptor across MDS/AML subtypes. To illustrate the therapeutic potential of CCRL2 as a therapeutic target, we developed an anti-CCRL2 antibody-drug conjugate (ADC) by conjugating an anti-CCRL2 antibody with the cytotoxic drug pyrrolobenzodiazepine (PBD), which causes DNA double-strand breaks leading to cancer cell death. The anti-CCRL2 ADC demonstrated strong CCRL2-selective cytotoxicity against cell lines derived from MDS/AML patients with TP53 mutations and erythroid features, surpassing the cytotoxic effects observed with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 ADCs. It also induced apoptosis and suppressed the clonogenicity of primary MDS/AML bone marrow samples without affecting the survival, differentiation and clonogenicity of healthy hematopoietic stem and progenitor cells. This agent also suppressed the leukemic growth of TP53-mutated MDS/AML cell line xenografts, improving mice survival and decreasing the leukemic burden in patient-derived TP53-mutated MDS/AML xenografts. In conclusion, our study introduces CCRL2 as a potential new therapeutic target in high-risk MDS/AML. Statement of significancePyrrolobenzodiazepine(PBD)-conjugated anti-CCRL2 ADC shows anti-leukemic effect in MDS/AML models including TP53-mutated disease without affecting healthy hematopoietic cells supporting that it is a promising candidate for single-agent or combination therapies in high-risk MDS/AML.

cancer biology↗

bioRxiv2025

CCRL2 promotes the interferon-γ signaling response in myeloid neoplasms with erythroid differentiation and mutated TP53

Patients with myeloid neoplasms with loss-of-function TP53 mutations and erythroid differentiation have poor outcomes, and a better understanding of disease biology is required. Upregulation of interferon-{gamma} (IFN-{gamma}) signaling has been associated with acute myeloid leukemia (AML) progression and chemotherapy resistance, but its drivers remain unclear. In this study, we found that the surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in AML with erythroid differentiation and TP53 mutations compared to other AML subtypes and healthy hematopoietic cells. The knockout (KO) of CCRL2 suppressed erythroleukemia growth in vitro and in vivo. Further proteomics and transcriptomics analysis revealed IFN-{gamma} signaling response as the top CCRL2-regulated pathway in erythroleukemia. Our mechanistic studies support direct CCRL2 driven IFN-{gamma} signaling independent of exogenous IFN-{gamma}, through phosphorylation of STAT1, via JAK2-dependent and independent mechanisms. CCRL2/IFN-{gamma} signaling is upregulated in erythroid leukemias, and TP53 mutated AML without concurrent increase of IFN-{gamma} secretion in the bone marrow microenvironment and is directly induced by TP53 KO. Finally, CCRL2/IFN-{gamma} signaling is associated with the transformation of pre-leukemic single-hit TP53 clones to multi-hit TP53 mutated AML, increased resistance to venetoclax and worse survival in AML. Overall, our findings support that CCRL2 is an essential driver of cell-autonomous IFN-{gamma} signaling response in myeloid neoplasms with erythroid differentiation and TP53 mutations and highlight CCRL2 as a relevant novel target for these neoplasms. One Sentence SummaryCCRL2 is overexpressed in AML with loss-of-function TP53 mutations and erythroid differentiation and promotes IFN-{gamma} signaling response via a cell-intrinsic mechanism.

cancer biology↗

bioRxiv2024

Dnmt3a-mediated de novo methylation balances Th1 and Tfh memory cell plasticity and functionality

Following acute viral infection, naive CD4+ T cells differentiate into T follicular helper (Tfh) and T helper 1 (Th1) cells that generate long-lived memory cells. However, it is unclear how memory Tfh and Th1 cells maintain their lineage commitment. We demonstrate that Tfh and Th1 lineages acquire distinct Dnmt3a-dependent de novo DNA methylation programs that are preserved into memory. Dnmt3a deletion impairs lineage commitment and functionality of memory Th1 and Tfh cells, resulting in aberrant Runx1 upregulation that represses germinal center Tfh cell differentiation. In contrast, transient pharmacological DNA methyltransferase inhibition during priming impairs repression of Tfh-associated genes while properly silencing Runx1, and results in enhanced Tfh cell functionality in primary and secondary responses to viral infections. Together, these findings demonstrate that Dnmt3a-mediated epigenetic programing is required to enforce T helper lineage commitment and preserve Tfh and Th1-specific functions during the recall response to infection, and reveal novel strategies to improve long-lived adaptive immunity against infectious diseases. SUMMARYThis article demonstrates that Dnmt3a-dependent epigenetic programing regulates functionality and plasticity of Th1 and Tfh memory cells. Furthermore, early pharmacological inhibition of such programing enhances GC Tfh cell differentiation, suggesting novel strategies for modulating the immune response to viral infections.

immunology↗

bioRxiv2023

In vivo validation of late-onset Alzheimer's disease genetic risk factors

Structured AbstractO_ST_ABSIntroductionC_ST_ABSGenome-wide association studies have identified over 70 genetic loci associated with late-onset Alzheimers disease (LOAD), but few candidate polymorphisms have been functionally assessed for disease relevance and mechanism of action. MethodsCandidate genetic risk variants were informatically prioritized and individually engineered into a LOAD-sensitized mouse model that carries the AD risk variants APOE4 and Trem2*R47H. Potential disease relevance of each model was assessed by comparing brain transcriptomes measured with the Nanostring Mouse AD Panel at 4 and 12 months of age with human study cohorts. ResultsWe created new models for 11 coding and loss-of-function risk variants. Transcriptomic effects from multiple genetic variants recapitulated a variety of human gene expression patterns observed in LOAD study cohorts. Specific models matched to emerging molecular LOAD subtypes. DiscussionThese results provide an initial functionalization of 11 candidate risk variants and identify potential preclinical models for testing targeted therapeutics.

neuroscience↗

bioRxiv2021

Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Muller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.

neuroscience↗

bioRxiv2021

The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia

The identification of new molecular pathways supporting the growth of myelodysplastic syndrome (MDS) stem and progenitor cells is needed to understand clinical variation and develop targeted therapies. Within myeloid malignancies, men have worse outcomes compared to women, suggesting male sex hormone driven effects in malignant hematopoiesis. The androgen receptor promotes the expression of five granulocyte-colony factor receptor regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor that regulates cytokine signaling in differentiated granulocytes but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is upregulated in stem and progenitor cells from patients with MDS and secondary acute leukemia. CCRL2 knockdown suppressed the growth and clonogenicity of MDS92 and MDS-L cells in vitro and in vivo. Moreover, CCRL2 knockdown significantly suppressed the phosphorylation of JAK2, STAT3, and STAT5 in MDS cells. CCRL2 co-precipitated with JAK2 and its suppression decreased the interaction of JAK2 with STAT proteins. Cell lines expressing JAK2V617F showed less effect of CCRL2 knockdown on growth and clonogenicity compared to those expressing wild type. However, the selective JAK2 inhibitor fedratinib potentiated the effects of CCRL2 knockdown in MDS and leukemia cells expressing both wild type JAK2 and JAK2V617F. In conclusion, our results implicate CCRL2 as a mediator of MDS and secondary acute leukemia cell growth, in part through JAK2/STAT signaling.

cancer biology↗

bioRxiv2020

Mutations in BBS2 Cause Apparent Nonsyndromic Retinitis Pigmentosa

PurposeTo identify and functionally test the causative mutations in the BBS2 gene in a family presenting with retinitis pigmentosa and infertility and to generate a bbs2-/- mutant zebrafish. MethodsA female proband and her male sibling were clinically evaluated and genetic testing with targeted next-generation sequencing was performed. Mutations were verified by Sanger sequencing. Protein localization was examined by transient expression and immunocytochemistry in cultured HEK-293T cells. Mutations in the zebrafish bbs2 gene were generated by CRISPR/Cas9 and retinal phenotypes were examined by immunohistochemistry. ResultsThe proband and her brother exhibited reduced visual fields, retinal degeneration, and bone spicule deposits, consistent with retinitis pigmentosa. The brother also reported symptoms consistent with infertility. Compound heterozygous mutations in the BBS2 gene; namely NM_031885.4 (BBS2):c.823C>T (p.R275X) and NM_031885.4 (BBS2):c.401C>G (p.P134R), were identified in the proband and her brother. Both mutations interfered with ciliary localization of Bbs2 in cell culture. Mutation of the zebrafish bbs2 gene resulted in progressive cone degeneration and rhodopsin mislocalization. ConclusionMissense mutations of BBS2 leads to non-syndromic retinitis pigmentosa, but not Bardet-Biedl Syndrome, even though Bbs2 fails to localize to cilia. In zebrafish, the complete loss of bbs2 results in cone degeneration and ciliopathy phenotypes, indicating a requirement for Bbs2 in photoreceptor survival.

genetics↗

bioRxiv2019

Primary cilia are present on endothelial cells of the hyaloid vasculature but are not required for the development of the blood-retinal barrier

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.

cell biology↗

bioRxiv2019

Ciliary Genes arl13b, ahi1 and cc2d2a Differentially Modify Expression of Visual Acuity Phenotypes but do not Enhance Retinal Degeneration due to Mutation of cep290 in Zebrafish

Mutations in the gene Centrosomal Protein 290 kDa (CEP290) result in multiple ciliopathies ranging from the neonatal lethal disorder Meckel-Gruber Syndrome to multi-systemic disorders such as Joubert Syndrome and Bardet-Biedl Syndrome to nonsyndromic diseases like Leber Congenital Amaurosis (LCA) and retinitis pigmentosa. Results from model organisms and human genetics studies, have suggest that mutations in genes encoding protein components of the transition zone (TZ) and other cilia-associated proteins can function as genetic modifiers and be a source for CEP290 pleiotropy. We investigated the zebrafish cep290fh297/fh297 mutant, which encodes a nonsense mutation (p.Q1217*). This mutant is viable as adults, exhibits scoliosis, and undergoes a slow, progressive cone degeneration. The cep290fh297/fh297 mutants showed partial mislocalization of the transmembrane protein rhodopsin but not of the prenylated proteins rhodopsin kinase (GRK1) or the rod transducin subunit GNB1. Surprisingly, photoreceptor degeneration did not trigger proliferation of Muller glia, but proliferation of rod progenitors in the outer nuclear layer was significantly increased. To determine if heterozygous mutations in other cilia genes could exacerbate retinal degeneration, we bred cep290fh297/fh297 mutants to arl13b, ahi1, and cc2d2a mutant zebrafish lines. While cep290fh297/fh297 mutants lacking a single allele of these genes did not exhibit accelerated photoreceptor degeneration, loss of one alleles of arl13b or ahi1 reduced visual performance in optokinetic response assays at 5 days post fertilization. Our results indicate that the cep290fh297/fh297 mutant is a useful model to study the role of genetic modifiers on photoreceptor degeneration in zebrafish and to explore how progressive photoreceptor degeneration influences regeneration in adult zebrafish.\n\nNonstandard abbreviations

cell biology↗

bioRxiv2018

Pregnancy associated plasma protein-aa (Pappaa) regulates photoreceptor synaptic development to mediate visually guided behavior

To guide behavior, sensory systems detect the onset and offset of stimuli and process these distinct inputs via parallel pathways. In the retina, this strategy is implemented by splitting neural signals for light onset and offset via synapses connecting photoreceptors to ON and OFF bipolar cells, respectively. It remains poorly understood which molecular cues establish the architecture of this synaptic configuration to split light onset and offset signals. A mutant with reduced synapses between photoreceptors and one bipolar cell type, but not the other, could reveal a critical cue. From this approach, we report a novel synaptic role pregnancy associated plasma protein aa (pappaa) in promoting the structure and function of cone synapses that transmit light offset information. Electrophysiological and behavioral analyses indicated pappaa mutant zebrafish have dysfunctional cone to OFF bipolar cell synapses and impaired responses to light offset, but intact cone to ON bipolar cell synapses and light onset responses. Ultrastructural analyses of pappaa mutant cones showed a lack of presynaptic domains at synapses with OFF bipolar cells. pappaa is expressed postsynaptically to the cones during retinal synaptogenesis and encodes a secreted metalloprotease known to stimulate insulin-like growth factor 1 (IGF1) signaling. Induction of dominant negative IGF1 receptor expression during synaptogenesis reduced light offset responses. Conversely, stimulating IGF1 signaling at this time improved pappaa mutants light offset responses and cone presynaptic structures. Together, our results indicate Pappaa-regulated IGF1 signaling as a novel pathway that establishes how cone synapses convey light offset signals to guide behavior.\n\nSignificance StatementDistinct sensory inputs, like stimulus onset and offset, are often split at distinct synapses into parallel circuits for processing. In the retina, photoreceptors and ON and OFF bipolar cells form discrete synapses to split neural signals coding light onset and offset, respectively. The molecular cues that establish this synaptic configuration to specifically convey light onset or offset remain unclear. Our work reveals a novel cue: pregnancy associated plasma protein aa (pappaa), which regulates photoreceptor synaptic structure and function to specifically transmit light offset information. Pappaa is a metalloprotease that stimulates local insulin-like growth factor 1 (IGF1) signaling. IGF1 promotes various aspects of synaptic development and function and is broadly expressed; thus requiring local regulators, like Pappaa, to govern its specificity.

neuroscience↗