Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo transport. Their future application is, however, hindered by the low efficiency of drug encapsulation and their poor adaptability in physiological conditions. To address these challenges, one potential solution is to incorporate micromotors with biological materials as the combination of functional biological entities and smart artificial parts represents a manipulable and biologically friendly approach. This dissertation focuses on the development of custom-designed micromotors combined with sperm and their potential applications on targeted diseases treatment. By means of 2D and 3D lithography methods, microstructures with complex configurations can be fabricated for specific demands. Bovine and human sperm are both for the first time explored as drug carriers thanks to their high encapsulation efficiency of hydrophilic drugs, their powerful self-propulsion and their improved drug-uptake relying on the somatic-cell fusion ability. The hybrid micromotors containing drug loaded sperm and constructed artificial enhancements can be self-propelled by the sperm flagella and remotely guided and released to the target at high precision by employing weak external magnetic fields. As a result, micromotors based on both bovine and human sperm show significant anticancer effect. The application here can be further broadened to other biological environments, in particular to the blood stream, showing the potential on the treatment of blood diseases like blood clotting. Finally, to enhance the treatment efficiency, in particular to control sperm number and drug dose, three strategies are demonstrated to transport swarms of sperm. This research paves the way for the precision medicine based on engineered sperm-based micromotors.

Indiana University-Purdue University Indianapolis (IUPUI) / Internal tandem duplications in FMS-like receptor tyrosine kinase (FLT3-ITDs) are seen in approximately 25% of all acute myeloid leukemia (AML) patients. FLT3-ITDs induce FLT3 ligand (FL)-independent cellular hyperproliferation, promiscuous and aberrant activation of STAT5, and confer a poor prognosis in patients; however, the molecular mechanisms contributing to FLT3-ITD-induced malignancy remain largely unknown. The protein tyrosine phosphatase, Shp2, is important for normal hematopoiesis as well as hematopoietic stem cell (HSC) differentiation, engraftment, and self-renewal. Furthermore, FLT3-ITD- or constitutive active STAT5-expressing CD34+ cells demonstrate enhanced hematopoietic stem cell self-renewal. Together with the previous findings that Shp2 is critical for normal hematopoiesis, that dysregulated Shp2 function contributes to myeloid malignancies, and that Shp2 has been shown to interact with WT-FLT3 tyrosine 599, which is commonly duplicated in FLT3-ITDs, a positive role for Shp2 in FLT3-ITD-induced signaling and leukemogenesis is implied. I demonstrated that Shp2 is constitutively associated with the reported FLT3-ITDs, N51-FLT3 and N73-FLT3, compared to WT-FLT3; therefore, I hypothesized that increased Shp2 recruitment to N51-FLT3 or N73-FLT3 contributes to hyperproliferation and hyperactivation of STAT5. I also hypothesized that Shp2 cooperates with STAT5 to activate STAT5 transcriptional targets contributing to the up-regulation of pro-leukemic proteins. Finally, I hypothesized that reduction of Shp2 would result in diminished N51-FLT3-induced hyperproliferation and activation of STAT5 in vitro, and prevent FLT3-ITD-induced malignancy in vivo. I found that genetic disruption of Ptpn11, the gene encoding Shp2, or pharmacologic inhibition of Shp2 with the novel Shp2 inhibitor, II-B08, resulted in significantly reduced FLT3-ITD-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation. I also demonstrated a novel role of Shp2 in the nucleus of FLT3-ITD-expressing hematopoietic cells where Shp2 and STAT5 co-localized at the promoter region of STAT5-transcriptional target and pro-survival protein, Bcl-XL. Furthermore, using a Shp2flox/flox;Mx1Cre+ mouse model, I demonstrated that reduced Shp2 expression in hematopoietic cells resulted in an increased latency to and reduced severity of FLT3-ITD-induced malignancy. Collectively, these findings demonstrate that Shp2 plays an integral role in FLT3-ITD-induced malignancy and suggest that targeting Shp2 may be a future therapeutic option for treating FLT3-ITD-positive AML patients.

Indiana University-Purdue University Indianapolis (IUPUI) / Graft-versus-host disease (GVHD) remains the major complication after allogeneic hematopoietic stem cell transplantation (HSCT), resulting from immunological attack on target organs such as gastrointestinal (GI) tract, liver and skin from donor allogeneic T cells. The most common treatment for GVHD is immunosuppressive drugs such as corticosteroids, which may result in many side effects including the loss of the beneficial graft-versus-leukemia (GVL) effect and increased infection rates. However, GVHD-specific drugs have yet to be implemented. Here we show that by targeting on a novel pathogenic CD4+ T cell subpopulation that our lab previously found in patients with GI GVHD, we can develop new avenues to treat GVHD. This novel population is characterized as CD146+CCR5+ T cells, co-expressing IL-17A and IFN-γ. We found that the inducible T-cell costimulator (ICOS), which has been reported to be important for human Th17 differentiation in vitro, is critical for the development of this nonconventional T Helper 1 (Th1*)-polarized CD146+CCR5+ conventional T cells (Tconvs) population. Furthermore, we found that ICOS can induce the generation of Th1*-polarized CD146+CCR5+ regulatory T cells (Tregs) population, lowering the frequencies of phenotypic markers of functional Tregs. Our data also showed that inhibiting the major transcriptional factor of Th17, RAR-related orphan receptor gamma t (RORγt), could prevent the development of CD146+CCR5+ Tconvs in vitro. Our results demonstrate how pathogenic CD146+CCR5+ T cells are induced through ICOS or RORγt, suggesting new targets for GVHD treatment. We anticipate our assay to be a starting point for the development of novel GVHD-specific drugs. For example, the treatments that focus on inhibiting RORγ would have fewer side effects than general immunosuppressive drugs that GVHD patients use today and inhibit GVHD while sparing the GVL effect. Furthermore, we expect the CD146+CCR5+ Tconvs and/or Tregs can be used as GVHD biomarkers. These biomarkers may guide preemptive treatments such as RORγt inhibitor.

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.