Katelyn J. Hoff's research while affiliated with University of Colorado and other places

Simple Summary Thyroid cancer is the most common endocrine malignancy. Patients with advanced thyroid cancers have poor survival rates, largely because of limited therapeutic options to combat their aggressive nature, creating a compelling need to identify novel therapeutic targets. We and others have previously shown that Src is a clinically relevant target in thyroid cancer and that combined inhibition of Src and the MAP kinase pathway results in enhanced anti-tumor responses. The goals of this study were to identify the mechanism(s) mediating these anti-tumor effects and identify additional potential biomarkers of response to improve therapies for patients with advanced thyroid cancer. Abstract Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

Heterozygous, missense mutations in both α- and β-tubulin genes have been linked to an array of neurodevelopment disorders, commonly referred to as “tubulinopathies.” To date, tubulinopathy mutations have been identified in three β-tubulin isotypes and one α-tubulin isotype. These mutations occur throughout the different genetic domains and protein structures of these tubulin isotypes, and the field is working to address how this molecular-level diversity results in different cellular and tissue-level pathologies. Studies from many groups have focused on elucidating the consequences of individual mutations; however, the field lacks comprehensive models for the molecular etiology of different types of tubulinopathies, presenting a major gap in diagnosis and treatment. This review highlights recent advances in understanding tubulin structural dynamics, the roles microtubule-associated proteins (MAPs) play in microtubule regulation, and how these are inextricably linked. We emphasize the value of investigating interactions between tubulin structures, microtubules, and MAPs to understand and predict the impact of tubulinopathy mutations at the cell and tissue levels. Microtubule regulation is multifaceted and provides a complex set of controls for generating a functional cytoskeleton at the right place and right time during neurodevelopment. Understanding how tubulinopathy mutations disrupt distinct subsets of those controls, and how that ultimately disrupts neurodevelopment, will be important for establishing mechanistic themes among tubulinopathies that may lead to insights in other neurodevelopment disorders and normal neurodevelopment.

Heterozygous, missense mutations in a- or b-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A -V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A -V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A -V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in mammalian cells have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which leads to α-tubulin antibody promiscuity and makes genetic manipulation challenging. Here, we test mutant tubulin levels and assembly activity and analyze the impact of TUBA1A reduction on growth cone composition, neurite extension, and commissural axon architecture during brain development. We present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduces TUBA1A protein levels and prevents incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice leads to grossly normal brain formation except a significant impact on axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a as a result of the Tuba1aND mutation exhibit slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting stabilization of microtubules. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration and cortex development but not commissure formation, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether the impact of a mutation at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8⁺ T cell recruitment and activation and a concomitant decrease in CD4⁺ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.