Lung adenocarcinoma (LUAD) is a highly heterogeneous disease with recurrent driver mutations. Given the recent increase in targeted therapies, many of these are now considered "actionable" mutations, yet clinical trials continue to show limited improvements in patient overall survival. Thus, there is an urgent need to identify the key pathways and regulators underlying tumor progression and resistance. Here, we investigate the role of Notch3, a known regulator of epithelial lung development and previously implicated in tumor-propagation capacity, in the lung adenocarcinoma context. We demonstrate that, while Notch activity is not required for cell survival, Kras-mutant LUAD cells undergo epithelial-to-mesenchymal transitional state changes in response both direct and indirect mechanisms engaged by transcriptionally active Notch3. We use transcriptomic and epigenetic analyses to define a set of Notch3 target genes and utilize genome-wide screening approaches to show that a subset of these regulate Notch3-induced therapeutic resistance to covalent Kras(G12C) inhibition. Given recent clinical data from patients and preclinical in vitro models supporting a role for EMT in Kras(G12C) resistance, our datasets provide unique epigenetic and transcriptional resources to discover potential therapeutic targets for overcoming resistance and tumor progression.

Noonan syndrome is a common dominant disorder characterized by short stature, facial dysmorphism, cardiac defects, and a predisposition to juvenile myelomonocytic leukemia (JMML). Germline PTPN11 mutations cause ~50% of Noonan syndrome. PTPN11 encodes SHP-2, a protein tyrosine phosphatase that relays signals from activated receptor complexes to Ras and other effectors. Studies of patient samples and in mouse models have demonstrated that hyperactive Ras signaling plays a central role in JMML. Based on the association of JMML with Noonan syndrome and the known role of SHP-2 in Ras signaling, our laboratory and others screened the PTPN11 gene in JMML, and discovered somatic mutations in ~ 35% of cases.

I performed functional studies of mutant SHP-2 proteins associated with JMML and Noonan syndrome. I used primary murine hematopoietic cells to investigate the effects of mutant SHP-2 on proliferation, survival, and differentiation. The most common leukemia-associated amino acid substitution (E76K) induced a hypersensitive pattern of myeloid progenitor colony growth in response to granulocyte-macrophage colony-stimulating factor and interleukin 3 that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells, perturbed erythroid growth, and impaired normal differentiation. In addition, leukemia-associated SHP-2 mutations conferred stronger phenotypes in primary hematopoietic progenitors than a germline mutation found in individuals with Noonan syndrome. Although PTPN11 mutations account for 50% of Noonan syndrome, the genetic lesions in the remaining individuals were unknown. Our collaborators discovered de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with Noonan syndrome and P34R and F156L alterations in individuals with cardio-facio-cutaneous syndrome, which has overlapping phenotypic features with Noonan syndrome. I performed biochemical and functional analysis of these novel syndrome-associated K-Ras proteins. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase activating proteins, and guanine nucleotide exchange all regulate developmental programs in vivo.

Early adenoviral proteins perturb the same pathways that are deregulated in cancer cells in order to drive viral replication. For example, E1A binds and inactivates the tumor suppressor pRb, while E1B-55K in conjunction with E4-ORF6 results in the degradation of p53. As a result, the studies of E1A and E1B have contributed greatly to our understanding of the Rb and p53 pathways, respectively. Other cellular pathways are also targeted by adenoviral proteins. Our lab has demonstrated that the early adenoviral protein E4-ORF1 potently activates PI3K alpha signaling. This activation is not through protein-protein interactions with PI3K enzymes, impinging on insulin signaling, disruption of Pten function, or activation of Ras family proteins. To determine how E4-ORF1 was activating the PI3K pathway, we used an unbiased proteomics approach to identify novel binding partners that played a role in PI3K signaling. We found the E4-ORF1 bound to a PDZ complex consisting of Cask, Mint1 and Veli-3 through another PDZ protein called Dlg1. Our results suggest that the Cask, Mint1, and Veli-3 complex acts as a repressor for PI3K signaling by both E4-ORF1 and growth factors, whereas Dlg1 is required for activation. E4-ORF1 and growth factors through an unknown cellular factor may be mislocalizing the repressor complex using Dlg1, which allows PI3K signaling to occur. Thus, the role of Dlg1, Cask, Mint1, and Veli-3 represents a novel regulatory step in this critical signaling pathway.

Mutations that deregulate proliferation and survival pathways have emerged as a common molecular theme in the pathogenesis of myeloproliferative disorders (MPDs). Three studies now report an amino acid substitution in the JAK2 kinase in most patients with polycythemia vera as well as in some cases of essential thrombocythemia and chronic idiopathic myelofibrosis. Functional analysis demonstrates that this mutation confers erythropoietin-independent growth in vitro, deregulates signaling pathways downstream of JAK2, and causes polycythemia in mice. These results open new avenues for diagnosing and classifying patients with these disorders, and identify a new molecular target for drug discovery.

Mutations that deregulate proliferation and survival pathways have emerged as a common molecular theme in the pathogenesis of myeloproliferative disorders (MPDs). Three studies now report an amino acid substitution in the JAK2 kinase in most patients with polycythemia vera as well as in some cases of essential thrombocythemia and chronic idiopathic myelofibrosis. Functional analysis demonstrates that this mutation confers erythropoietin-independent growth in vitro, deregulates signaling pathways downstream of JAK2, and causes polycythemia in mice. These results open new avenues for diagnosing and classifying patients with these disorders, and identify a new molecular target for drug discovery.

Targeted therapy has achieved clinical success in many cancer patients whose diseases are driven by defined genomic lesions resulting in pathologically activated kinases. While mutations revealed by tumor sequencing can identify novel therapeutic targets, it is critical to distinguish between non-transforming “passenger” mutations and transforming “driver” mutations potentially amenable to therapeutic targeting. In contrast with oncogenic ABL1 fusions, point mutations in the ABL1 proto-oncogene have yet to be validated for transforming capacity and potential for therapeutic targeting. We assessed a number of ABL1 1b point mutants identified in clinical isolates and found that select mutations transformed Ba/F3 cells to growth factor independence and caused constitutive kinase activation. Employing an in vitro random mutagenesis screen, we prospectively identified additional activating mutants that could be vulnerable to currently available ABL1 inhibitors. All activating ABL1 1b mutants characterized in our study exhibited sensitivity to ATP-competitive tyrosine kinase inhibitors and relative resistance to allosteric inhibitors targeting the myristoyl-binding pocket regardless of proximity to the allosteric site. As the allosteric inhibitor ABL001 is currently undergoing clinical trial evaluation for treatment-refractory chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoid leukemia, we cloned these ABL1 1b-activating mutations into BCR-ABL1 and found that most, except Y469H and P918L, also caused significant resistance to allosteric inhibitors. Thus, we identified BCR-ABL1 mutations with the potential to promote clinical resistance to an emerging class of allosteric inhibitors. Finally, we assessed cDNA from a cohort of CML patients expressing a dominant BCR-ABL1 T315I mutation, and targeted sequencing of ABL1 1b revealed a heterozygous T315I mutation in one patient. Presently, CML patients who acquire resistance to ATP-competitive ABL1 TKIs are screened only for BCR-ABL1 mutations, but our finding that select mutations confer greater resistance in the context of ABL1 1b suggests drug-resistant patients should be assessed for mutations in both ABL1 1b and BCR-ABL1. Another challenge to developing effective therapies is that acquired drug resistance or toxicity from multi-kinase inhibition frequently limits the long-term efficacy of TKIs. Activating mutations of the FLT3 receptor tyrosine kinase are validated therapeutic targets in acute myeloid leukemia, but available FLT3 TKIs are susceptible to secondary resistance mutations and limited in clinical utility by off-target effects. We interrogated the role of dimerization in FLT3 activation to address whether inhibiting receptor dimerization could be a therapeutic target. Using FLT3 truncation constructs and phosphorylation site mutants, we unexpectedly found that the cytoplasmic domain of oncogenic FLT3 ITD is highly transforming and resistant to TKIs, likely through enhanced STAT5 signaling, implying the extracellular and/or transmembrane domains may have an autoinhibitory function. Thus, it will be important to evaluate the relative contributions of different FLT3 domains to activation, dimerization, and autoinhibition. A better understanding of cooperativity between modes of kinase activation and deregulation could aid in developing novel therapeutic strategies exploiting fundamental protein regulatory mechanisms.

Glucocorticoids (GCs) are potently immunosuppressive due to their pro-apoptotic effects on lymphoid cells, leading to their utility in a wide range of clinical contexts including autoimmune diseases, hyperinflammatory conditions, and lymphoid malignancies. In addition, GCs are endogenously produced hormones that mediate a wide range of physiologic functions that are essential for life. As a result, cells of the immune system face constant exposure to GCs and therefore require intrinsic mechanisms by which to resist their pro-apoptotic effects under certain developmental and environmental conditions. The retention of these resistance mechanisms in lymphoid-driven diseases thereby poses a challenge to the clinical use of GCs in these contexts. Common -chain cytokines, through activation of the JAK/STAT signal transduction pathway, play essential roles in the differentiation, survival, and function of lymphoid cells. Here, we assess the interplay between GC activity and cytokine-mediated pro-survival signaling in two lymphoid-driven diseases, acute lymphoblastic leukemia and hemophagocytic lymphohistiocytosis, and demonstrate that cytokines are potent mediators of GC resistance. Mechanistically, we show that activation of STAT5 downstream of cytokine receptor signaling is necessary for cytokine-induced GC resistance. STAT5 functions as a transcription factor to upregulate expression of anti-apoptotic mediators, thereby reducing the apoptotic potential and promoting the survival of lymphoid cells. Through both genetic and pharmacologic approaches, we demonstrate that targeted inhibition of cytokine receptor signaling effectively restores GC sensitivity, and we establish the feasibility of this therapeutic approach in two in vivo models of disease.