Developing novel therapeutic strategies for the treatment of TET2-mutated acute myeloid leukaemia

Ph. D. Thesis.Acute myeloid leukaemia (AML) is a clinically heterogeneous disease driven by somatic mutations and chromosomal abnormalities. TET2 mutations occur in 7%-23% of AML, with approximately 75% monoallelic and 25% biallelic mutations. TET2 encodes a protein involved in regulating DNA demethylation and TET2-mutated AML has a significantly lower event-free survival and higher relapse rate compared to disease with wild-type TET2. As such, there is an urgent clinical need to develop novel methods to treat patients with TET2-mutated AML. Hypomethylating agents such as azacytidine that inhibits DNA methylation are now established as a therapeutic strategy for AML. This study evolved from an index patient with biallelic TET2-mutated AML, that was chemoresistant to anthracycline and cytarabine, but acutely sensitive to azacytidine monotherapy resulting in long-term morphological remission with overall survival of 850 days. The primary focus of my research is to interrogate the role of TET2 mutant allele dosage in determining AML cell response to azacytidine. Using an isogenic cell model system, we demonstrated that biallelic TET2 mutation leads to complete loss of TET2 protein and acquisition of a hypermethylated phenotype. Of note, TET2 mutant allele dosage significantly affected the azacytidine sensitivity of cells. TET2 biallelic mutants were hypersensitive to azacytidine with significantly lower proliferation in liquid media (P<0.0001) and cloning efficiency (P=0.0056) compared to their isogenic TET2 monoallelic mutants. RNA-sequencing identified ABCB1, a major azacytidine efflux transporter, to be downregulated in cells with TET2 biallelic mutation. Inhibition of ABCB1 using selective inhibitors (Tariquidar and Verapamil) increased sensitivity to azacytidine. Also, azacytidine resistant AML cell clones were developed through long-term exposure of TET2 mutated cells to azacytidine. ABCB1 is upregulated in resistant clones compared to the azacytidine sensitive cells and inhibition of ABCB1 re-sensitised the resistant clones to azacytidine. In addition, gene ontology analysis identified ribosomal pathway components to be downregulated in TET2 biallelic mutated cells compared to their isogenic TET2 monoallelic mutated counterparts. As such, the findings of this investigation suggest the potential involvement of multiple genes and pathways in sensitizing TET2 null cells to azacytidine. Finally, using ten different AML cell lines, we demonstrated that there was significant correlation between TET2 protein expression and azacytidine IC90 (R2=0.77, P=0.0008) and IC50 (R2=0.88, P<0.0001). Together, our findings argue in favour of using azacytidine for the treatment of TET2 biallelic mutated AML and highlights the importance of TET2 mutant allele dosage in developing precision medicine in AML.Newcastle University Overseas Research Scholarship (NUORS), Funds for Women Graduates (FFWG), Bloodwis

Milled non-mulberry silk fibroin microparticles as biomaterial for biomedical applications

Silk fibroin has been widely employed in various forms as biomaterials for biomedical applications due to its superb biocompatibility and tunable degradation and mechanical properties. Herein, silk fibroin microparticles of non-mulberry silkworm species (Antheraea assamensis, Antheraea mylitta and Philosamia ricini) were fabricated via a top-down approach using a combination of wet-milling and spray drying techniques. Microparticles of mulberry silkworm (Bombyx mori) were also utilized for comparative studies. The fabricated microparticles were physico-chemically characterized for size, stability, morphology, chemical composition and thermal properties. The silk fibroin microparticles of all species were porous (&sim;5&mu;m in size) and showed nearly spherical morphology with rough surface as revealed from dynamic light scattering and microscopic studies. Non-mulberry silk microparticles maintained the typical silk-II structure with &beta;-sheet secondary conformation with higher thermal stability. Additionally, non-mulberry silk fibroin microparticles supported enhanced cell adhesion, spreading and viability of mouse fibroblasts than mulberry silk fibroin microparticles (p&lt;0.001) as evidenced from fluorescence microscopy and cytotoxicity studies. Furthermore, in vitro drug release from the microparticles showed a significantly sustained release over 3 weeks. Taken together, this study demonstrates promising attributes of non-mulberry silk fibroin microparticles as a potential drug delivery vehicle/micro carrier for diverse biomedical applications

Silk fibroin–keratin based 3D scaffolds as a dermal substitute for skin tissue engineering

Development of highly vascular dermal tissue-engineered skin substitutes with appropriate mechanical properties and cellular cues is in need for significant advancement in the field of dermal reconstruction. Limitations have been imposed on natural biomaterials despite their superb biocompatibility hence, studies in biomaterial blending have been ongoing. Herein, we investigated blends of silk fibroin and human hair-derived keratin as wound-healing substrates that promote enhanced fibroblast cell adhesion and proliferation. Three-dimensional (3D) blended scaffolds were fabricated by freeze-drying, and their physico-chemical, mechanical and degradable properties were extensively characterized. Cytocompatibility tests observing cell adhesion and cell proliferation have shown significant enhancements in blended scaffolds. Also, its structural composition with high porosity (>85%) and interconnected pores in the range of 100–120 microns further confirms the superiority of the complex compared to its counterparts. FTIR studies identified the enhanced stability within its structure and were followed-up with sequential experiments to demonstrate improved thermal, degradation, and mechanical properties. Furthermore, immunohistochemical staining revealed greater expression of collagen type I in the cultured cells, indicating functional fibroblast proliferation and, hence, the exciting potential of this construct for dermal applications. Taken together, this study demonstrates the promising attributes from blended biomaterials and specifically present silk fibroin and human hair keratin blended scaffolds as a promising dermal substitute for skin tissue engineering.NRF (Natl Research Foundation, S’pore)NMRC (Natl Medical Research Council, S’pore

Role of green tea catechins in modulating stromal-epithelial interaction in prostate cells

28-36Recent years have witnessed a significant interest in phytonutrients as treatment module for a range of diseases, including cancers. Catechins, predominant in green tea are potent antioxidants belonging to the flavonoid family that have been linked epidemiologically to a lower prevalence of prostate pathologies in Asian nations. In the present work, we investigated ameliorative potential of green tea catechins (epigallocatechin, EGC, and epigallocatechin 3-gallate, EGCG) on the aberrant proliferation of normal prostate epithelial cells driven by paracrine action of stromal medium, conditioned with estradiol (E2) and dihydrotestosterone (DHT) (20 pM and 10 nM respectively). It was observed that EGC (5 μM) significantly inhibited the aberrant epithelial proliferation induced by stromal conditioned medium (CM), with a decreased expression of ERα, ERβ, AR, CK05, CK18, and PSA. Further, the CM collected after pre-treatment of stromal cells with EGCG (0.75 μM), had an inhibitory effect on epithelial cell proliferation, with reduction in the expression of ERα, AR, and CK05 while the expression of ERβ, CK18, and PSA were increased. These findings suggest that EGC helps in downregulating the aberrant epithelial proliferation while EGCG is able to downregulate the proliferative potential of stromal cell secretome. These insights will help in deciphering preventive measures against prostate pathologies involving stromal-epithelial cross-talk

Biallelic TET2 mutation sensitizes to 5’-azacitidine in acute myeloid leukemia

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5'-azacitidine (5'-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5'-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5'-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer

Biallelic TET2 mutations confer sensitivity to 5 '-azacitidine in acute myeloid leukemia

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5 '-azacitidine (5 '-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5 '-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5 '-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer