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.

Mesenchymal stem cells promote metastasis through activation of an ABL-MMP9 signaling axis in lung cancer cells.

Mesenchymal stem cells (MSCs) are recruited and activated by solid tumors and play a role in tumor progression and metastasis. Here we show that MSCs promote metastasis in a panel of non-small cell lung cancer (NSCLC) cells. MSCs elicit transcriptional alterations in lung cancer cells leading to increased expression of factors implicated in the epithelial-to-mesenchymal transition (EMT) and secreted proteins including matrix metalloproteinase-9 (MMP9). MSCs enhance secretion of enzymatically active MMP9 in a panel of lung adenocarcinoma cells. High expression of MMP9 is linked to low survival rates in lung adenocarcinoma patients. Notably, we found that ABL tyrosine kinases are activated in MSC-primed lung cancer cells and functional ABL kinases are required for MSC-induced MMP9 expression, secretion and proteolytic activity. Importantly, ABL kinases are required for MSC-induced NSCLC metastasis. These data reveal an actionable target for inhibiting MSC-induced metastatic activity of lung adenocarcinoma cells through disruption of an ABL kinase-MMP9 signaling axis activated in MSC-primed lung cancer cells.

Correction: ABL kinase inhibition sensitizes primary lung adenocarcinomas to chemotherapy by promoting tumor cell differentiation.

[This corrects the article DOI: 10.18632/oncotarget.26740.].

ABL kinase inhibition sensitizes primary lung adenocarcinomas to chemotherapy by promoting tumor cell differentiation.

Lung cancer is the leading cause of cancer mortality in the United States, with an overall five-year survival rate of ~16%. Non-small cell lung cancer (NSCLC) accounts for ~80% of all lung cancer cases, and the majority (40%) of these are adenocarcinomas. Loss of function point mutations in TP53 (46%) and activating mutations in KRAS (33%) are the most common mutations in human lung adenocarcinomas. Because neither of these genetic alterations are clinically actionable, chemotherapy remains the mainstay of treatment in patients with oncogenic KRAS driver mutations. However, chemoresistance to genotoxic agents such as docetaxel remains a major clinical challenge facing lung cancer patients. Here we show that ABL kinase allosteric inhibitors can be effectively used for the treatment of KrasG12D/+; p53-/- lung adenocarcinomas in an autochthonous mouse model. Unexpectedly, we found that treatment of tumor-bearing mice with an ABL allosteric inhibitor promoted differentiation of lung adenocarcinomas from poorly differentiated tumors expressing basal cell markers to tumors expressing terminal differentiation markers in vivo, which rendered lung adenocarcinomas susceptible to chemotherapy. These findings uncover a novel therapeutic approach for the treatment of lung adenocarcinomas with poor response to chemotherapy.

Inactivation of ABL kinases suppresses non-small cell lung cancer metastasis.

Current therapies to treat non-small cell lung carcinoma (NSCLC) have proven ineffective owing to transient, variable, and incomplete responses. Here we show that ABL kinases, ABL1 and ABL2, promote metastasis of lung cancer cells harboring EGFR or KRAS mutations. Inactivation of ABL kinases suppresses NSCLC metastasis to brain and bone, and other organs. ABL kinases are required for expression of prometastasis genes. Notably, ABL1 and ABL2 depletion impairs extravasation of lung adenocarcinoma cells into the lung parenchyma. We found that ABL-mediated activation of the TAZ and ß-catenin transcriptional coactivators is required for NSCLC metastasis. ABL kinases activate TAZ and ß-catenin by decreasing their interaction with the ß-TrCP ubiquitin ligase, leading to increased protein stability. High-level expression of ABL1, ABL2, and a subset of ABL-dependent TAZ- and ß-catenin-target genes correlates with shortened survival of lung adenocarcinoma patients. Thus, ABL-specific allosteric inhibitors might be effective to treat metastatic lung cancer with an activated ABL pathway signature.

Abl family kinases modulate T cell-mediated inflammation and chemokine-induced migration through the adaptor HEF1 and the GTPase Rap1.

Chemokine signaling is critical for T cell function during homeostasis and inflammation and directs T cell polarity and migration through the activation of specific intracellular pathways. Here, we uncovered a previously uncharacterized role for the Abl family tyrosine kinases Abl and Arg in the regulation of T cell-dependent inflammatory responses and showed that the Abl family kinases were required for chemokine-induced T cell polarization and migration. Our data demonstrated that Abl and Arg were activated downstream of chemokine receptors and mediated the chemokine-induced tyrosine phosphorylation of human enhancer of filamentation 1 (HEF1), an adaptor protein that is required for the activity of the guanosine triphosphatase Rap1, which mediates cell adhesion and migration. Phosphorylation of HEF1 by Abl family kinases and activation of Rap1 were required for chemokine-induced T cell migration. Mouse T cells that lacked Abl and Arg exhibited defective homing to lymph nodes and impaired migration to sites of inflammation. These findings suggest that Abl family kinases are potential therapeutic targets for the treatment of T cell-dependent immune disorders that are characterized by chemokine-mediated inflammation.

Abl tyrosine kinases in T-cell signaling.

Stimulation of the T-cell antigen receptor (TCR) leads to the activation of signaling pathways that are essential for T-cell development and the response of mature T cells to antigens. The TCR has no intrinsic catalytic activity, but TCR engagement results in tyrosine phosphorylation of downstream targets by non-receptor tyrosine kinases. Three families of tyrosine kinases have long been recognized to play critical roles in TCR-dependent signaling. They are the Src, zeta-associated protein of 70 kDa, and Tec families of kinases. More recently, the Abelson (Abl) tyrosine kinases have been shown to be activated by TCR engagement and to be required for maximal TCR signaling. Using T-cell conditional knockout mice deficient for Abl family kinases, Abl (Abl1) and Abl-related gene (Arg) (Abl2), it was recently shown that loss of Abl kinases results in defective T-cell development and a partial block in the transition to the CD4(+)CD8(+) stage. Abl/Arg double null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Moreover, conditional knockout mice lacking Abl and Arg in T cells exhibit impaired CD8(+) T-cell expansion in vivo upon Listeria monocytogenes infection. Thus, Abl kinase signaling is required for both T-cell development and mature T-cell function.

Defective T cell development and function in the absence of Abelson kinases.

Thymocyte proliferation, survival, and differentiation are tightly controlled by signaling from the pre-TCR. In this study, we show for the first time that the Abelson (Abl) kinases regulate proximal signaling downstream of the pre-TCR. Conditional deletion of Abl kinases in thymocytes reveals a cell-autonomous role for these proteins in T cell development. The conditional knockout mice have reduced numbers of thymocytes, exhibit an increase in the percentage of the CD4(-)CD8(-) double-negative population, and are partially blocked in the transition to the CD4(+)CD8(+) double-positive stage. Moreover, the total number of T cells is greatly reduced in the Abl mutant mice, and the null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Notably, Abl mutant mice are compromised in their ability to produce IFN-positive CD8 T cells and exhibit impaired CD8(+) T cell expansion in vivo upon Listeria monocytogenes infection. Furthermore, Ab production in response to T cell-dependent Ag is severely impaired in the Abl mutant mice. Together these findings reveal cell-autonomous roles for the Abl family kinases in both T cell development and mature T cell function, and show that loss of these kinases specifically in T cells results in compromised immunity.

Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling.

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.

Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl.

Bcr-Abl tyrosine kinase activity initiates a number of intracellular signaling cascades that result in leukemogenesis. Imatinib mesylate, a specific Bcr-Abl tyrosine kinase inhibitor, has been highly successful in the treatment of chronic myelogenous leukemia (CML). However, the emergence of imatinib resistance and the incomplete molecular response of a significant number of patients receiving this therapy have led to a search for combinations of drugs that will enhance the efficacy of imatinib. We have demonstrated that mycophenolic acid (MPA), a specific inosine monophosphate dehydrogenase (IMPDH) inhibitor that results in depletion of intracellular guanine nucleotides, is synergistic with imatinib in inducing apoptosis in Bcr-Abl-expressing cell lines. Studies of signaling pathways downstream of Bcr-Abl demonstrated that the addition of MPA to imatinib reduced the phosphorylation of both Stat5 and Lyn, a Src kinase family member. The phosphorylation of S6 ribosomal protein was also greatly reduced. These results demonstrate that inhibitors of guanine nucleotide biosynthesis may synergize with imatinib in reducing the levels of minimal residual disease in CML and lay the foundation for clinical trials in which IMPDH inhibitors are added to imatinib in patients who have suboptimal molecular responses to single agent therapy or who have progressive disease.

On the molecular pathology of neurodegeneration in IMPDH1-based retinitis pigmentosa.

Retinitis pigmentosa (RP), the hereditary degenerative disease of the photoreceptor neurons of the retina, probably represents the most prevalent cause of registered blindness amongst those of working age in developed countries. Mutations within the gene encoding inosine monophosphate dehydrogenase 1 (IMPDH1), the widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have recently been shown to cause the RP10 form of autosomal dominant RP. We examined the expression of IMPDH1, IMPDH2 and HPRT transcripts, encoding enzymes of the de novo and salvage pathways of guanine nucleotide biosynthesis, respectively, in retinal sections of mice, the data indicating that the bulk of GTP within photoreceptors is generated by IMPDH1. Impdh1(-/-) null mice are shown here to display a slowly progressive form of retinal degeneration in which visual transduction, analysed by electroretinographic wave functions, becomes gradually compromised, although at 12 months of age most photoreceptors remain structurally intact. In contrast, the human form of RP caused by mutations within the IMPDH1 gene is a severe autosomal dominant degenerative retinopathy in those families that have been examined to date. Expression of mutant IMPDH1 proteins in bacterial and mammalian cells, together with computational simulations, indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity, is the likely cause of the severe phenotype experienced by human subjects. Taken together, these findings suggest that RP10 may represent an attractive target for therapeutic intervention, based upon a strategy combining simultaneous suppression of transcripts from normal and mutant IMPDH1 alleles with supplementation of GTP within retinal tissues.

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to xanthosine monophosphate (XMP) at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides. Inhibition of IMPDH results in the depletion of guanine nucleotides, prevents cell growth by G1 arrest, and induces cell differentiation in a cell-type-specific manner. The molecular and sensing mechanisms underlying these effects are not clear. We have examined the induction of apoptosis by mycophenolic acid (MPA), a specific IMPDH inhibitor, in interleukin-3 (IL-3)-dependent murine hematopoietic cell lines. MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3. Apoptosis was completely prevented by the addition of guanosine at time points up to 12 hours, after which caspase 3 activity increased and apoptosis was not reversible. MPA treatment caused marked down-regulation of the MAP kinase kinase/extracellular regulatory kinase (MEK/Erk) pathway at 3 hours while simultaneously increasing the phosphorylation of c-Jun kinase. In addition, MPA strongly down-regulated the mammalian target of rapamcyin (mTOR) pathway, as indicated by the decreased phosphorylation of p70 S6 kinase and of 4EBP1. Inhibition of either the mitogen-activated protein kinase (MAPK) or the mTOR pathway alone by standard pharmacologic inhibitors did not induce apoptosis in IL-3-dependent cells, whereas inhibition of both pathways simulated the effects of MPA treatment. These results indicate that IMPDH inhibitors may be effective in modulating signal transduction pathways in hematopoietic cells, suggesting their usefulness in chemotherapeutic regimens for hematologic malignancies.

Inhibition of nucleoside transport by p38 MAPK inhibitors.

While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.