ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

The ABL2 kinase regulates an HSF1-dependent transcriptional program required for lung adenocarcinoma brain metastasis.

Brain metastases are the most common intracranial tumors in adults and are associated with increased patient morbidity and mortality. Limited therapeutic options are currently available for the treatment of brain metastasis. Here, we report on the discovery of an actionable signaling pathway utilized by metastatic tumor cells whereby the transcriptional regulator Heat Shock Factor 1 (HSF1) drives a transcriptional program, divergent from its canonical role as the master regulator of the heat shock response, leading to enhanced expression of a subset of E2F transcription factor family gene targets. We find that HSF1 is required for survival and outgrowth by metastatic lung cancer cells in the brain parenchyma. Further, we identify the ABL2 tyrosine kinase as an upstream regulator of HSF1 protein expression and show that the Src-homology 3 (SH3) domain of ABL2 directly interacts with HSF1 protein at a noncanonical, proline-independent SH3 interaction motif. Pharmacologic inhibition of the ABL2 kinase using small molecule allosteric inhibitors, but not ATP-competitive inhibitors, disrupts this interaction. Importantly, knockdown as well as pharmacologic inhibition of ABL2 using allosteric inhibitors impairs expression of HSF1 protein and HSF1-E2F transcriptional gene targets. Collectively, these findings reveal a targetable ABL2-HSF1-E2F signaling pathway required for survival by brain-metastatic tumor cells.

Pharmacology and in vivo efficacy of pyridine-pyrimidine amides that inhibit microtubule polymerization.

Microtubule-targeting agents are important tools in cancer treatment. Generating novel microtubule targeting agents with novel pharmacology could dramatically expand the utility of this class of drugs. Here we characterize the pharmacology of recently described small molecule microtubule polymerization inhibitors. Pharmacokinetic experiments show oral bioavailability through gastric absorption. In vitro assays designed to predict absorption, distribution, metabolism, and excretion (ADME) and safety reveal a scaffold that is metabolically stable, evades P-glycoprotein, does not inhibit CYP enzymes, occurs as a significant free fraction in serum, and has exceptionally high cellular permeability. Together with in vivo efficacy models, pharmacology supports further development as a treatment for solid tumors.

Pyridine-pyrimidine amides that prevent HGF-induced epithelial scattering by two distinct mechanisms.

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure-activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer.

Cellular contractility changes are sufficient to drive epithelial scattering.

Epithelial scattering occurs when cells disassemble cell-cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial-mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell-cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell-cell interactions occurs by remodeling of cell-cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell-cell adhesion between cells in suspension, suggesting that changes in cell-cell adhesion strength might not accompany epithelial scattering. Instead, cell-substrate adhesion seems to play a bigger role, as cell-substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFß is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells׳ ability to scatter. Further consistent with changes in substrate adhesion being required for cell-cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell-cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell-cell detachment during epithelial scattering.

CBF-Beta Mitigates PI3K-Alpha-Specific Inhibitor Killing through PIM1 in PIK3CA-Mutant Gastric Cancer.

PIK3CA is the second most mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased translation, proliferation, and survival. Some 4%-25% of gastric cancers display activating PIK3CA mutations, including 80% of Epstein-Barr virus-associated GCs. Small molecules, including pan-PI3K and dual PI3K/mTOR inhibitors, have shown moderate success clinically, due to broad on-target/off-tissue effects. Thus, isoform-specific and mutant selective inhibitors have been of significant interest. However, drug resistance is a problem and has affected success of new drugs. There has been a concerted effort to define mechanisms of resistance and identify potent combinations in many tumor types, though gastric cancer is comparatively understudied. In this study, we identified modulators of the response to the PI3Kα-specific inhibitor, BYL719, in PIK3CA-mutant GCs. We found that loss of NEDD9 or inhibition of BCL-XL conferred hypersensitivity to BYL719, through increased cell-cycle arrest and cell death, respectively. In addition, we discovered that loss of CBFB conferred resistance to BYL719. CBFB loss led to upregulation of the protein kinase PIM1, which can phosphorylate and activate several overlapping downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3Kα inhibition. The addition of a pan-PIM inhibitor re-sensitized resistant cells to BYL719. Our data provide clear mechanistic insights into PI3Kα inhibitor response in PIK3CA-mutant gastric tumors and can inform future work as mutant-selective inhibitors are in development for diverse tumor types. IMPLICATIONS: Loss of either NEDD9 or BCL-XL confers hypersensitivity to PI3K-alpha inhibition whereas loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.

Executioner caspases restrict mitochondrial RNA-driven Type I IFN induction during chemotherapy-induced apoptosis.

During apoptosis, mitochondrial outer membrane permeabilization (MOMP) enables certain mitochondrial matrix macromolecules to escape into the cytosol. However, the fate of mitochondrial RNA (mtRNA) during apoptosis is unknown. Here, we demonstrate that MOMP results in the cytoplasmic release of mtRNA and that executioner caspases-3 and -7 (casp3/7) prevent cytoplasmic mtRNA from triggering inflammatory signaling. In the setting of genetic or pharmacological casp3/7 inhibition, apoptotic insults result in mtRNA activation of the MDA5/MAVS/IRF3 pathway to drive Type I interferon (IFN) signaling. This pathway is sufficient to activate tumor-intrinsic Type I IFN signaling in immunologically cold cancer models that lack an intact cGAS/STING signaling pathway, promote CD8+ T-cell-dependent anti-tumor immunity, and overcome anti-PD1 refractoriness in vivo. Thus, a key function of casp3/7 is to inhibit inflammation caused by the cytoplasmic release of mtRNA, and pharmacological modulation of this pathway increases the immunogenicity of chemotherapy-induced apoptosis.

Activation of KrasG12D in Subset of Alveolar Type II Cells Enhances Cellular Plasticity in Lung Adenocarcinoma.

We have previously identified alveolar type II cell as the cell-of-origin of KrasG12D-induced lung adenocarcinoma using cell lineage-specific inducible Cre mouse models. Using gain-of-function and loss-of-function genetic models, we discovered that active Notch signaling and low Sox2 levels dictate the ability of type II cells to proliferate and progress into lung adenocarcinoma upon KrasG12D activation. Here, we examine the phenotype of type II cells after Kras activation and find evidence for proliferation of cells that coexpress type I and type II markers. Three-dimensional organoid culture and transplantation studies determine that these dual-positive cells are highly plastic and tumor initiating in vivo. RNA sequencing analysis reveals that these dual-positive cells are enriched in Ras/MAPK, EGFR, and Notch pathways. Furthermore, the proliferation of these cells requires active Notch signaling and is inhibited by genetic/chemical Sox2 upregulation. Our findings could provide new therapeutic strategies to target KRAS-activated lung adenocarcinomas. SIGNIFICANCE: Identification of progenitor like tumor-initiating cells in KRAS-mutant lung adenocarcinoma may allow development of novel targeted therapeutics.

MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

ABL allosteric inhibitors synergize with statins to enhance apoptosis of metastatic lung cancer cells.

Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease.

A TAZ-AXL-ABL2 Feed-Forward Signaling Axis Promotes Lung Adenocarcinoma Brain Metastasis.

Brain metastases are a common consequence of advanced lung cancer, resulting in cranial neuropathies and increased mortality. Currently, there are no effective therapies to treat brain metastases due to a lack of actionable targets and a failure of systemic therapies to penetrate the blood-brain barrier (BBB). Here we identify an autocrine signaling axis required for lung adenocarcinoma brain metastasis, whereby nuclear accumulation of the TAZ transcriptional co-activator drives expression of a panel of transcripts enriched in brain metastases, including ABL2 and AXL, encoding for protein tyrosine kinases that engage in bidirectional signaling. Activation of ABL2 in turn promotes TAZ tyrosine phosphorylation and nuclear localization, establishing an autocrine AXL-ABL2-TAZ feed-forward signaling loop required for brain metastasis colonization. Notably, treatment with a BBB-penetrant ABL allosteric inhibitor or knockdown of ABL2, AXL, or TAZ markedly decreases brain metastases. These findings suggest that ABL and AXL inhibitors might be effective against brain metastases.