Biology subjects

Ghiaur, G.

Publications and source records attributed to Ghiaur, G..

7 recordsLinked to original sources

bioRxiv2026

Modular Assembly of an Infectious Clone of the Akata strain of EBV using Synthetic Genomics Methods in Yeast

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Synthetic Biology↗

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

Serine auxotrophy is a targetable vulnerability driven by PSAT1 suppression in AML

Serine metabolism is of growing biologic and therapeutic interest in cancer. Upregulation of the serine synthesis pathway (SSP) can fuel tumor growth, and cancers with this phenotype are often sensitive to SSP inhibitors. In parallel, dietary restriction of serine and glycine (SG) can suppress some cancers, but the determinants of sensitivity to this approach are poorly understood. This is especially true in acute myeloid leukemia (AML), where serine metabolism has been less explored. We report that a subset of human AML cell lines and primary samples are completely dependent on external serine, known as serine auxotrophy. These leukemias consistently suppressed the SSP enzyme PSAT1, failed to synthesize serine, responded to SG restriction in vivo, and were rescued by restoring PSAT1. We also found that AML with an SF3B1 K700E mutation showed additional dependence on the SSP enzyme PHGDH, that SG restriction synergized with venetoclax in serine auxotrophic AML, and that MECOM rearrangement was strongly associated with PSAT1 suppression and serine auxotrophy. These findings define a metabolically distinct AML subtype and nominate it for targeting by SG restriction.

cancer biology↗

bioRxiv2023

A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

PurposeThe limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, we developed [68Ga]Ga-AJ206, a peptide-based radiotracer that can be seamlessly integrated into the standard clinical workflow and is specifically designed to non-invasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). Experimental designWe synthesized a high-affinity binder for quantification of CD38 levels. Affinity was tested using surface plasmon resonance, and In vitro specificity was evaluated using a gallium-68-labeled analog. Distribution, pharmacokinetics, and CD38 specificity of the radiotracer were assessed in MM cell lines and in primary patient-derived myeloma cells and xenografts (PDX) with cross-validation by flow cytometry and immunohistochemistry. Furthermore, we investigated the radiotracers potential to quantify CD38 pharmacodynamics induced by all-trans retinoic acid therapy (ATRA). Results[68Ga]Ga-AJ206 exhibited high CD38 binding specificity (KD: 19.1{+/-}0.99 nM) and CD38-dependent In vitro binding. [68Ga]Ga-AJ206-PET showed high contrast within 60 minutes and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detected CD38 expression in xenografts, PDXs and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantified CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following ATRA therapy. Conclusions[68Ga]Ga-AJ206 exhibited the salient features required for clinical translation, providing CD38-specific high contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement. STATEMENT OF TRANSLATIONAL RELEVANCEThere is an unmet need for functional imaging agents to monitor the pharmacodynamic effects of new therapeutics targeting multiple myeloma (MM). MM is a challenging bone marrow plasma cell cancer that is associated with heterogenous responses and universal recurrence. While minimal residual disease monitoring by blood and invasive bone marrow samples have improved prognostication of disease recurrence, molecularly targeted, non-invasive imaging options that can assess therapy response early remain limited. To address this gap, we report the development of a high affinity, first-in-class gallium-68 labeled peptide radiotracer, [68Ga]Ga-AJ206, for CD38 protein, which is highly expressed on MM cells. [68Ga]Ga-AJ206 provides high-contrast CD38-specific images by PET within the standard clinical workflow of 60 minutes. Furthermore, the potential of [68Ga]Ga-AJ206 PET to measure pharmacodynamics of CD38 was demonstrated. Further development of this new radiotracer may complement existing technologies and improve prognostication and monitoring of therapy response in patients with MM.

cancer biology↗

bioRxiv2022

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAVs. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.

molecular biology↗

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↗

bioRxiv2018

DNMT3A-NPM1 mutated acute myeloid leukaemia shows sensitivity to a PARP1 inhibitor combined with daunorubicin in an in vitro model

Acute myeloid leukaemia is a neoplasia in need of new treatment approaches. PARP inhibitors are a class of targeted therapeutics for cancer that disrupts dysfunctional DNA damage response in various neoplasia. MLL-AF9 mutated leukaemias are sensitive to combinations of PARP inhibitors and cytotoxic drugs. Moreover, DNMT3A and NPM1 mutations are linked to dysfunctions in DNA damage response. Therefore, we investigated if DNMT3A-NPM1 mutated AML cell line is sensible to PARP inhibitors combined with anthracyclines. Our results show that DNMT3A-NPM1 mutated AML is as sensible to combinations of PARP inhibitors and anthracyclines as MLL-AF9 mutated leukaemias, in an in vitro setting.

cancer biology↗