261 research outputs found
A Synthetic Lethal shRNA Screen and Genetic Proof of Concept Identifies RAC1 as a Novel Target to Disrupt Plexiform Neurofibroma Formation
No full textIndiana University-Purdue University Indianapolis (IUPUI)Neurofibromatosis Type 1 (NF1) is a highly penetrant autosomal dominant
genetic disorder where mutations in the tumor suppressor gene NF1 leads to decreased
neurofibromin. The most debilitating manifestation is the presence of complex multilineage
Schwann cell-derived plexiform neurofibromas (PN). Historically, little clinical
success has been achieved targeting PN through surgery or chemotherapies. I performed
an shRNA library screen of patient-derived Schwann cell lines to identify novel
therapeutic targets to disrupt PN formation and progression. An shRNA library screen of
human kinases and Rho-GTPases was performed in NF1-/- and paired NF1 competent
immortalized Schwann cell lines. Following sequencing, candidates were identified. We
previously developed a novel mouse model of NF1 wherein a neural crest specific Postncre
targeted loxp-flanked Nf1 that replicated the PN found in patients. Additional cohorts
of mice were generated with biallelic deletion of Rac1 (Nf1f/fRac1f/f Postn-Cre+; DKO ).
Mice were aged for 9 months and peripheral nerves were harvested and fixed in formalin.
Peripheral nerve size was measured and tumors were identified through blinded analysis
of hematoxylin and eosin and Masson’s Trichrome (collagen) stained slides. Rho family
members, including RAC1, were identified as candidates through an shRNA library
screen. Genetic disruption of Rac1 in the Schwann cell lineage resulted in the prevention
of tumor formation in DKO mice, as observed by peripheral nerve size and histological
analysis. I observed an average of 14.8 +/- 2.65 tumors per mouse in the Nf1f/f Postnviii
Cre+ cohort compared to 0 tumors in the DKO (p<0.0001). Following an shRNA library
screen, RAC1 was identified as a candidate to modulate PN formation. Biallelic deletion
of Rac1 in vivo prevented PN formation. I demonstrate that a candidate identified in an
shRNA library screen can translate to an biological effect in a mouse model of PN
Abstract IA24: Identification of therapies for plexiform neurofibromas, a precursor of malignant peripheral nerve sheath tumors
No full textA Mechanistic Approach to Identify Novel Therapeutic Drugs for Targeting FA-Disrupted Malignancies
No full textIndiana University-Purdue University Indianapolis (IUPUI)The Fanconi anemia (FA) signaling network plays a critical role in maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in Fanconi Anemia, a cancer predisposition syndrome characterized by congenital malformations, bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are found in 30% of all sporadic cancers and over 15% of sporadic pediatric AMLs underscoring the importance of this pathway in the prevention of malignant transformation. Therefore, the identification of precision therapies for FA-deficient AML is a critical need. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established. We and others have uncovered the roles of FA proteins in mitotic regulation, suggesting additional mechanisms by which the FA pathway prevents genomic instability. Mutation of FANCA is the most common cause of FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. To identify synthetic lethal targets of FANCA, we previously identified mitotic phospho-signaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. We identified mitotic kinases CHEK1, PLK1, SLK, and TTK as potential targets, which suggests a mitosis-specific vulnerability of FA-deficient cells. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway members, FANCG and BRCA1, suggesting that inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. A more thorough understanding of FA pathway function in mitosis provides new insight into AML pathogenesis and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies
A Mechanistic Approach to Identify Novel Therapeutic Drugs for Targeting FA-Disrupted Malignancies
No full textIndiana University-Purdue University Indianapolis (IUPUI)The Fanconi anemia (FA) signaling network plays a critical role in maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in Fanconi Anemia, a cancer predisposition syndrome characterized by congenital malformations, bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are found in 30% of all sporadic cancers and over 15% of sporadic pediatric AMLs underscoring the importance of this pathway in the prevention of malignant transformation. Therefore, the identification of precision therapies for FA-deficient AML is a critical need. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established. We and others have uncovered the roles of FA proteins in mitotic regulation, suggesting additional mechanisms by which the FA pathway prevents genomic instability. Mutation of FANCA is the most common cause of FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. To identify synthetic lethal targets of FANCA, we previously identified mitotic phospho-signaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. We identified mitotic kinases CHEK1, PLK1, SLK, and TTK as potential targets, which suggests a mitosis-specific vulnerability of FA-deficient cells. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway members, FANCG and BRCA1, suggesting that inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. A more thorough understanding of FA pathway function in mitosis provides new insight into AML pathogenesis and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies
Expression of Human Papillomavirus Type 16 E7 Is Sufficient To Significantly Increase Expression of Angiogenic Factors But Is Not Sufficient To Induce Endothelial Cell Migration
No full textIndiana University-Purdue University Indianapolis (IUPUI)Human papillomavirus 16 (HPV 16) causes cancer. Two viral oncoproteins of HPV 16, E6 and E7, are consistently expressed in these cancers. HPV 16 E6 and E7 proteins target p53 and Rb family members, respectively, for degradation thus inactivating the functiond of these tumor suppressor proteins. Tumor development requires the acquisition of a blood supply, a process known as angiogenesis. Tumor suppressors negatively regulate angigogenesis. Expression of HPV 16 E6 and E7 together in human foreskin keratinocytes (HFKs) increases the level of angiogenic inducers vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Further, conditioned media from such cells are sufficient to alter endothelial cell behavior both in vitro and in vivo. To determine the individual contributions of HPV E6 and E7 to angiogenesis, translational termination linkers (TTLs) were inserted into the coding region of E6 or E7. Following retroviral transduction of the mutated cassette into HFKs, the ability of E7 in the context of the E6TTL mutation (E6TTLE7) and E6 in the context of the E7TTL mutation (E6E7TTL) to induce VEGF and IL-8 was compared to the LXSN control retrovirus. E7 and, to a lesser extent E6, increased the expression of VEGF and IL-8. Migration of human microvascular endothelial cells was not induced using conditioned media from either E6 or E7 expressing cells. Since the increased levels of VEGF and IL-8 induced by HPV 16E6ETTLE7 were not sufficient to alter endothelial cell behavior, immunological depletion experiments were used to determine whether either angiogenic factor was required for HPV 16E6 and E7 together to induce HMVEC migration. Only VEGF was required. Preliminary data suggest that the ability of HPV 16 E7 to induce angiogenic factors is dependent upon degradation of a specific Rb family member
IN VIVO HEMATOPOIETIC CELL ENGRAFTMENT IS MODULATED BY DPPIV/CD26 INHIBITION AND RHEB2 OVEREXPRESSION
No full textIndiana University-Purdue University Indianapolis (IUPUI)Hematopoietic cell transplantation (HCT) is an important modality used to treat patients with hematologic diseases and malignancies. A better understanding of the biological processes controlling hematopoietic cell functions such as migration/homing, proliferation and self-renewal is required for improving HCT therapies. This study focused on the role of two biologically relevant proteins, dipeptidylpeptidase IV (DPPIV/CD26) and Ras homologue enriched in brain 2 (Rheb2), in modulating hematopoietic cell engraftment. The first goal of this study was to determine the role of the protein DPPIV/CD26 in modulating the engraftment of human umbilical cord blood (hUCB) CD34+ stem/progenitor cells using a NOD/SCID mouse xenograft model, and based upon previous work demonstrating a role for this enzyme in Stromal-Derived Factor-1/CXCL12 mediated migration and homing. Related to this first goal, pretreatment with an inhibitor of DPPIV/CD26 peptidase activity increased engraftment of hUCB CD34+ cells in vivo in recipient Non Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice while not disturbing their differentiation potential following transplantation. These results support using DPPIV/CD26 inhibition as a strategy for enhancing the efficacy of cord blood transplantation. The second goal was to determine, by overexpression, the role of the Rheb2 in affecting the balance between proliferation and in vivo repopulating activity of mouse hematopoietic cells. Rheb2 is known to activate the mammalian target of rapamycin (mTOR) pathway, a pathway important in hematopoiesis. Rheb2 overexpression increased the proliferation and mTOR signaling of two hematopoietic cell lines, 32D and BaF3, in response to delayed IL-3 addition. In primary mouse hematopoietic cells, Rheb2 overexpression enhanced the proliferation and expansion of hematopoietic progenitor cells (HPCs) and phenotypic hematopoietic stem cells (HSCs) in vitro. In addition, HPC survival was enhanced by Rheb2 overexpression. Using in vivo competitive repopulation assays, Rheb2 overexpression transiently expanded immature HPC/HSC populations shortly after transplantation, but reduced the engraftment of total transduced cells. These findings support previous work showing that signaling proteins able to enhance the proliferative status of hematopoietic stem cells often cause exhaustion of self-renewal and repopulating ability. These studies of hematopoietic engraftment modulated by both of these molecules provide information which may be important to future work on HCT
Fanconi anemia and the cell cycle: new perspectives on aneuploidy
No full textFanconi anemia (FA) is a complex heterogenic disorder of genomic instability, bone marrow failure, cancer predisposition, and congenital malformations. The FA signaling network orchestrates the DNA damage recognition and repair in interphase as well as proper execution of mitosis. Loss of FA signaling causes chromosome instability by weakening the spindle assembly checkpoint, disrupting centrosome maintenance, disturbing resolution of ultrafine anaphase bridges, and dysregulating cytokinesis. Thus, the FA genes function as guardians of genome stability throughout the cell cycle. This review discusses recent advances in diagnosis and clinical management of Fanconi anemia and presents the new insights into the origins of genomic instability in FA. These new discoveries may facilitate the development of rational therapeutic strategies for FA and for FA-deficient malignancies in the general population
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