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Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti-resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin-related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAF V600E melanomas. In clinical trials, pretreatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG-257081 and anti-PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG-257081 can improve the response to immune checkpoint blockade. CCG-257081 reduced the expression of PDL1 in BRAFi-resistant melanoma cells and decreased surface PDL1 levels on both BRAFi-sensitive and -resistant melanoma cells. Using our recently described murine vemurafenib-resistant melanoma model, we found that CCG-257081, in combination with anti-PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti-PD1/CCG-257081 co-treatment increased infiltration of CD8 + T cells and B cells into the tumor microenvironment and reduced tumor-associated macrophages. Here, we propose CCG-257081 as an anti-resistance and immune therapy-enhancing anti-melanoma agent., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

The oxidative balance of a cell is maintained by the Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway. This cytoprotective pathway detoxifies reactive oxygen species and xenobiotics. The role of the KEAP1/NRF2 pathway as pro-tumorigenic or anti-tumorigenic throughout stages of carcinogenesis (including initiation, promotion, progression, and metastasis) is complex. This mini review focuses on key studies describing how the KEAP1/NRF2 pathway affects cancer at different phases. The data compiled suggest that the roles of KEAP1/NRF2 in cancer are highly dependent on context; specifically, the model used (carcinogen-induced vs genetic), the tumor type, and the stage of cancer. Moreover, emerging data suggests that KEAP1/NRF2 is also important for regulating the tumor microenvironment and how its effects are amplified either by epigenetics or in response to co-occurring mutations. Further elucidation of the complexity of this pathway is needed in order to develop novel pharmacological tools and drugs to improve patient outcomes.

NRF2 is a transcription factor that controls the cellular response to various stressors, such as reactive oxygen and nitrogen species. As such, it plays a key role in the suppression of carcinogenesis, but constitutive NRF2 expression in cancer cells leads to resistance to chemotherapeutics and promotes metastasis. As a result, inhibition of the NRF2 pathway is a target for new drugs, especially for use in conjunction with established chemotherapeutic agents like carboplatin and 5-fluorouracil. A new class of NRF2 inhibitors has been discovered with substituted nicotinonitriles, such as MSU38225. In this work, the effects on NRF2 inhibition with structural changes were explored. Through these studies, we identified a few compounds with as good or better activity than the initial hit but with greatly improved solubility. The syntheses involved a variety of metal-catalyzed reactions, including titanium multicomponent coupling reactions and various Pd and Cu coupling reactions. In addition to inhibiting NRF2 activity, these new compounds inhibited the proliferation and migration of lung cancer cells in which the NRF2 pathway is constitutively activated., Competing Interests: A patent application has been submitted on the compounds in this article., (This journal is © The Royal Society of Chemistry.)

Single agent and combination therapy with BRAF V600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Misek, Foda, Dexheimer, Akram, Conrad, Schmidt, Neubig and Gallo.)

Most B-Raf proto-oncogene (BRAF)-mutant melanoma tumors respond initially to BRAF inhibitor (BRAFi)/mitogen-activated protein kinase kinase 1 inhibitor (MEKi) therapy, although few patients have durable long-term responses to these agents. The goal of this study was to use an unbiased computational approach to identify inhibitors that reverse an experimentally derived BRAFi resistance gene expression signature. Using this approach, we found that ibrutinib effectively reverses this signature, and we demonstrate experimentally that ibrutinib resensitizes a subset of BRAFi-resistant melanoma cells to vemurafenib. Ibrutinib is used clinically as an inhibitor of the Src family kinase Bruton tyrosine kinase (BTK); however, neither BTK deletion nor treatment with acalabrutinib, another BTK inhibitor with reduced off-target activity, resensitized cells to vemurafenib. These data suggest that ibrutinib acts through a BTK-independent mechanism in vemurafenib resensitization. To better understand this mechanism, we analyzed the transcriptional profile of ibrutinib-treated BRAFi-resistant melanoma cells and found that the transcriptional profile of ibrutinib was highly similar to that of multiple Src proto-oncogene kinase inhibitors. Since ibrutinib, but not acalabrutinib, has appreciable off-target activity against multiple Src family kinases, it suggests that ibrutinib may be acting through this mechanism. Furthermore, genes that are differentially expressed in ibrutinib-treated cells are enriched in Yes1-associated transcriptional regulator (YAP1) target genes, and we showed that ibrutinib, but not acalabrutinib, reduces YAP1 activity in BRAFi-resistant melanoma cells. Taken together, these data suggest that ibrutinib, or other Src family kinase inhibitors, may be useful for treating some BRAFi/MEKi-refractory melanoma tumors. SIGNIFICANCE STATEMENT: MAPK-targeted therapies provide dramatic initial responses, but resistance develops rapidly; a subset of these tumors may be rendered sensitive again by treatment with an approved Src family kinase inhibitor-ibrutinub-potentially providing improved clinical outcomes., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAF V600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAF V600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

Acquired resistance to doxorubicin is a major hurdle in triple-negative breast cancer (TNBC) therapy, emphasizing the need to identify improved strategies. Apigenin and other structurally related dietary flavones are emerging as potential chemo-sensitizers, but their effect on three-dimensional TNBC spheroid models has not been investigated. We previously showed that apigenin associates with heterogeneous ribonuclear protein A2/B1 (hnRNPA2), an RNA-binding protein involved in mRNA and co-transcriptional regulation. However, the role of hnRNPA2 in apigenin chemo-sensitizing activity has not been investigated. Here, we show that apigenin induced apoptosis in TNBC spheroids more effectively than apigenin-glycoside, owing to higher cellular uptake. Moreover, apigenin inhibited the growth of TNBC patient-derived organoids at an in vivo achievable concentration. Apigenin sensitized spheroids to doxorubicin-induced DNA damage, triggering caspase-9-mediated intrinsic apoptotic pathway and caspase-3 activity. Silencing of hnRNPA2 decreased apigenin-induced sensitization to doxorubicin in spheroids by diminishing apoptosis and partly abrogated apigenin-mediated reduction of ABCC4 and ABCG2 efflux transporters. Together these findings provide novel insights into the critical role of hnRNPA2 in mediating apigenin-induced sensitization of TNBC spheroids to doxorubicin by increasing the expression of efflux transporters and apoptosis, underscoring the relevance of using dietary compounds as a chemotherapeutic adjuvant., (Copyright © 2020. Published by Elsevier Inc.)

Dysregulation of intracellular signaling pathways is a key attribute of diseases associated with chronic inflammation, including cancer. Mitogen activated protein kinases have emerged as critical conduits of intracellular signal transmission, yet due to their ubiquitous roles in cellular processes, their direct inhibition may lead to undesired effects, thus limiting their usefulness as therapeutic targets. Mixed lineage kinases (MLKs) are mitogen-activated protein kinase kinase kinases (MAP3Ks) that interact with scaffolding proteins and function upstream of p38, JNK, ERK, and NF-kappaB to mediate diverse cellular signals. Studies involving gene silencing, genetically engineered mouse models, and small molecule inhibitors suggest that MLKs are critical in tumor progression as well as in inflammatory processes. Recent advances indicate that they may be useful targets in some types of cancer and in diseases driven by chronic inflammation including neurodegenerative diseases and metabolic diseases such as nonalcoholic steatohepatitis. This review describes existing MLK inhibitors, the roles of MLKs in various aspects of tumor progression and in the control of inflammatory processes, and the potential for therapeutic targeting of MLKs., Competing Interests: Declaration of Competing Interest None, (Copyright © 2019. Published by Elsevier Inc.)

Over half of cutaneous melanoma tumors have BRAF V600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that ~50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anticancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the Rho High BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

In prior work we demonstrated that loss of E2F transcription factors inhibits metastasis. Here we address the mechanisms for this phenotype and identify the E2F regulated genes that coordinate tumor cell metastasis. Transcriptomic profiling of E2F1 knockout tumors identified a role for E2F1 as a master regulator of a suite of pro-metastatic genes, but also uncovered E2F1 target genes with an unknown role in pulmonary metastasis. High expression of one of these genes, Fgf13, is associated with early human breast cancer metastasis in a clinical dataset. Together these data led to the hypothesis that Fgf13 is critical for breast cancer metastasis, and that upregulation of Fgf13 may partially explain how E2F1 promotes breast cancer metastasis. To test this hypothesis we ablated Fgf13 via CRISPR. Deletion of Fgf13 in a MMTV-PyMT breast cancer cell line reduces colonization of the lungs in a tail vein injection. In addition, loss of Fgf13 reduced in vitro cell migration, suggesting that Fgf13 may be critical for tumor cells to escape the primary tumor and to colonize the distal sites. The significance of this work is twofold: we have both uncovered genomic features by which E2F1 regulates metastasis and we have identified new pro-metastatic functions for the E2F1 target gene Fgf13.

A hallmark of glioblastoma (GBM) tumors is their highly invasive behavior. Tumor dissemination into surrounding brain tissue is responsible for incomplete surgical resection, and subsequent tumor recurrence. Identification of targets that control GBM cell dissemination is critical for developing effective therapies to treat GBM. A majority of GBM tumors have dysregulated EGFR signaling, due most frequently to EGFR amplification or the presence of a constitutively active EGFRvIII mutant. Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that can activate multiple MAPK pathways. In this study, evidence is provided that MLK3 is essential for GBM cell migration and invasion, and that an MLK inhibitor blocks EGF-induced migration and invasion. MLK3 silencing or MLK inhibition blocks EGF-induced JNK activation, suggesting that MLK3-JNK signaling promotes invasion of GBM cells. Mechanistically, it is demonstrated that DOCK180, a RAC1 guanine nucleotide exchange factor (GEF) overexpressed in invasive GBM cells, activates the MLK3-JNK signaling axis in a RAC1-dependent manner. In summary, this investigation identifies an EGFR-DOCK180-RAC1-MLK3-JNK signaling axis that drives glioblastoma cell migration and dissemination. Implications: On the basis of these findings, MLK3 emerges as a potential therapeutic target for the treatment of glioblastoma. Mol Cancer Res; 15(8); 1085-95. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Mixed-lineage kinase 3 (MLK3), a mitogen-activated protein kinase kinase kinase (MAP3K), has critical roles in metastasis of triple-negative breast cancer (TNBC), in part by regulating paxillin phosphorylation and focal adhesion turnover. However the mechanisms and the distinct step(s) of the metastatic processes through which MLK3 exerts its influence are not fully understood. Here we report that in non-metastatic, estrogen receptor-positive breast cancer (ER+ BC) cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), which is accompanied by elevation of matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced ER+ BC cell invasion is abrogated by FRA-1 silencing, demonstrating that MLK3 drives invasion through FRA-1. Conversely, in metastatic TNBC models, high FRA-1 levels are significantly reduced upon depletion of MLK3 by either gene silencing or by the CRISPR/Cas9n editing approach. Furthermore, ablation of MLK3 or MLK inhibitor treatment decreases expression of both MMP-1 and MMP-9. Consistent with the role of tumor cell-derived MMP-1 in endothelial permeability and transendothelial migration, both of these are reduced in MLK3-depleted TNBC cells. In addition, MLK inhibitor treatment or MLK3 depletion, which downregulates MMP-9 expression, renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells derived from TNBC-bearing mice display increased levels of FRA-1 and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in vascular intravasation. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMPs axis suggest that MLK3 is a promising therapeutic target for treatment of TNBC.

Mixed-lineage kinase 3 (MLK3) was first cloned in 1994; however, only in the past decade has MLK3 become recognized as a player in oncogenic signaling. MLK3 is a mitogen-activated protein kinase kinase kinase (MAP3K) that mediates signals from several cell surface receptors including receptor tyrosine kinases (RTKs), chemokine receptors, and cytokine receptors. Once activated, MLK3 transduces signals to multiple downstream pathways, primarily to c-Jun terminal kinase (JNK) MAPK, as well as to extracellular-signal-regulated kinase (ERK) MAPK, P38 MAPK, and NF-κB, resulting in both transcriptional and post-translational regulation of multiple effector proteins. In several types of cancer, MLK3 signaling is implicated in promoting cell proliferation, as well as driving cell migration, invasion and metastasis.

Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO), are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER)-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864) and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer.

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.

Introduction. Breast cancer recurrence can develop years after primary treatment. Crosstalk between breast cancer cells and their stromal microenvironment may influence tumor progression. Our primary study aim was to determine whether endothelin-1 (ET-1) expression in tumor and stroma predicts breast cancer relapse. The secondary aim was to determine ET-1/endothelin receptor A (ETAR) role on signaling pathways and apoptosis in breast cancer. Experimental Design. Patients with histologically documented stages I-III invasive breast cancer were included in the study. ET-1 expression by immunohistochemistry (IHC) in tumor cells and stroma was analyzed. Association between ET-1 expression and clinical outcome was assessed using multivariate Cox proportional hazard model. Kaplan-Meier curves were used to estimate disease-free survival (DFS). In addition, the effect of ET-1/ETAR on signaling pathways and apoptosis was evaluated in MCF-7 and MDA-MB-231 breast cancer cells. Results. With a median followup of 7 years, ET-1 non-enriched tumor phenotype had a significant association with favorable disease-free survival (HR = 0.16; 95% CI 0.03-0.77; P value <0.02). ER negativity, advanced stage of disease and ET-1-enriched tumor phenotype were all associated with a higher risk for recurrence. Experimental study demonstrated that ET-1 stimulation promoted Akt activation in MCF-7 and MDA-MB-231 cells. Furthermore, silencing of ETAR induced apoptosis in both hormone receptor negative and hormone receptor positive breast cancer cells. Conclusions. We found ET-1 expression in tumor and stroma to be an independent prognostic marker for breast cancer recurrence. Prospective studies are warranted to examine whether ET-1 expression in tumor/stroma could assist in stratifying patients with hormone receptor positive breast cancer for adjuvant therapy.

Mixed-lineage kinase 3 (MLK3) activates multiple MAPK pathways and can initiate apoptosis, proliferation, migration, or differentiation in different cell types. However, whether MLK3 signaling regulates intestinal epithelial cell sheet migration in vivo is not known. We sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility in vivo and in vitro. In vivo, we compared the healing of jejunal mucosal ulcers induced in MLK3 knockout (KO) mice with healing in wild-type (WT) mice. Ulcer healing was 20.8% less at day 3 (P < 0.05) and 18.9% less at day 5 (P < 0.05) in MLK3 KO than WT mice. Within the intestinal mucosa of MLK3 KO mice, ERK and JNK signaling were reduced, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) level was increased, and p38 signaling was unchanged. Parallel in vitro studies using an MLK inhibitor assessed the role of MLK signaling in human Caco-2 intestinal epithelial migration across collagen substrates. The MLK inhibitor reduced closure of circular wounds in Caco-2 monolayers. MLK inhibition reduced ERK and JNK, but not p38, signaling in Caco-2 cells. Although PTEN is increased after MLK inhibition, it does not influence MLK-mediated cell migration. These findings indicate that disruption of MLK3 signaling impairs ulcer healing by suppressing ERK and JNK signaling in vitro and in mouse intestinal mucosa in vivo. These results reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration in vivo and suggest that MLK3 may be an important target for the regulation of intestinal mucosal healing.

MLK3 kinase activates multiple mitogen-activated protein kinases and plays a critical role in cancer cell migration and invasion. In the tumor microenvironment, prometastatic factors drive breast cancer invasion and metastasis, but their associated signaling pathways are not well-known. Here, we provide evidence that MLK3 is required for chemokine (CXCL12)-induced invasion of basal breast cancer cells. We found that MLK3 induced robust phosphorylation of the focal adhesion scaffold paxillin on Ser 178 and Tyr 118, which was blocked by silencing or inhibition of MLK3-JNK. Silencing or inhibition of MLK3, inhibition of JNK, or expression of paxillin S178A all led to enhanced Rho activity, indicating that the MLK3-JNK-paxillin axis limits Rho activity to promote focal adhesion turnover and migration. Consistent with this, MLK3 silencing increased focal adhesions and stress fibers in breast cancer cells. MLK3 silencing also decreased the formation of breast cancer lung metastases in vivo, and breast cancer cells derived from mouse lung metastases showed enhanced Ser 178 paxillin phosphorylation. Taken together, our findings suggest that the MLK3-JNK-paxillin signaling axis may represent a potential therapeutic target and/or prognostic marker in breast cancer metastasis., (©2012 AACR.)

The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3's catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3's contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3-JNK-AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFalpha (tumour necrosis factor alpha). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the 'MLK3 protease', reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

Some 5 years ago, it was first discovered that mutations in the gene encoding LRRK2 (leucine-rich repeat protein kinase 2) are tightly linked with a subset of familial PD (Parkinson's disease). Before this genetic association, LRRK2 had never been investigated biochemically. Now it is of utmost importance to establish whether LRRK2 is a bona fide kinase in vitro and in vivo and to understand how mutations of LRRK2 lead to the specific loss of dopaminergic neurons in the substantia nigra to cause PD. In spite of tremendous efforts in the research community, there is no consensus with regard to the magnitude of the enzymatic activity of LRRK2 mutant forms that segregate with PD owing, in part, to the lack of a highly sensitive kinase assay system, and it is still unclear whether an abnormal increase in kinase activity is responsible for LRRK2-associated PD. As described in this issue of the Biochemical Journal, Nichols et al.. have developed an extensive set of molecular tools, including an optimized peptide substrate for determining in vitro kinase activity of LRRK2, a set of kinase inhibitors that can be used to explore LRRK2 substrate specificity and biology, a much-needed murine-specific antibody for immunoprecipation, and efficient gene-silencing approaches. In the present commentary, we discuss some of the components of this new LRRK2 biochemical toolbox and how they can be used to better understand this enigmatic kinase.

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.

Parkinson's disease (PD) is the most common motor neurodegenerative disease. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been linked recently with autosomal-dominant parkinsonism that is clinically indistinguishable from typical, idiopathic, late-onset PD. Thus, the protein LRRK2 has emerged as a promising therapeutic target for treatment of PD. LRRK2 is extraordinarily large and complex, with multiple enzymatic and protein-interaction domains, each of which is targeted by pathogenic mutations in familial PD. This review places the PD-associated mutations of LRRK2 in a structural and functional framework, with the ultimate aim of deciphering the molecular basis of LRRK2-associated pathogenesis. This, in turn, should advance our understanding and treatment of familial and idiopathic PD.

Activation of c-jun N-terminal kinase (JNK) signaling is associated with neuronal cell death. As described in this issue of Chemistry & Biology, cell-based screening efforts yielded a compound, AEG3482, which interacts with heat shock protein 90 leading to inhibition of JNK and blockade of neuronal apoptosis .

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH(2)-terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG.MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90alpha,beta (Hsp90) and its kinase-specific co-chaperone p50(cdc37). We show that endogenous MLK3 complexes with Hsp90 and p50(cdc37). Further experiments demonstrate that MLK3 associates with Hsp90/p50(cdc37) through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor alpha-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50(cdc37) regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50(cdc37) clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50(cdc37) also regulates protein kinases involved in apoptotic signaling.

Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.

Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases.

MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42.

Mixed lineage kinase 3 (MLK 3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with MAPK pathways. The aim of this study was to test the potential interaction of MLK 3 with signaling pathways stimulated by PDGF in rat mesangial cells. We have established a stable cell line expressing human MLK 3 in rat glomerular mesangial cells. The effects of PDGF on proliferation and matrix mRNA expression were examined. In control (vector-transfected) mesangial cells PDGF increased [(3)H]-thymidine incorporation significantly in a concentration-dependent manner. In mesangial cells expressing MLK 3, PDGF-induced increase in DNA synthesis was significantly reduced. PDGF also induced fibronectin and collagen I mRNA expression in control cells, the effects of which were also significantly blocked in MLK 3-transfected cells. To understand the potential interaction of MLK 3 over expression with the MAPK pathways and to examine the potential mechanism of the effects of MLK 3 over expression on proliferation and matrix expression, activation of ERK2, JNK1 and p38 were examined. ERK2 activation was increased several fold by PDGF in control cells but was attenuated significantly in MLK 3 expressing cells. PDGF did not have any effect on JNK and p38 activation, in either cell types. Using the same stable-transfected cell line, identical results were obtained on proliferation and matrix expression with sarafotoxin-s6b (endothelin receptor agonist) another potent mitogenic and sclerotic agent for mesangial cells. These results indicate an important role for MLK 3 in the regulation of growth and matrix expression in mesangial cells., (Copyright 2002 S. Karger AG, Basel)

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.

Src homology 3 domain-containing proline-rich kinase (SPRK)/mixed lineage kinase-3 is a serine/threonine kinase that has been identified as an upstream activator of the c-Jun NH(2)-terminal kinase (JNK) pathway. SPRK is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites Ser(254) and Thr(258) block SPRK-induced JNK activation. A region of 63 amino acids following the kinase domain of SPRK is predicted to form a leucine zipper. The leucine zipper domain of SPRK has been shown to be necessary and sufficient for SPRK oligomerization, but its role in regulating activation of SPRK and downstream signaling remains unclear. In this study, we substituted a proposed stabilizing leucine residue in the zipper domain with a helix-disrupting proline to abrogate zipper-mediated SPRK oligomerization. We demonstrate that constitutively activated Cdc42 fully activates this monomeric SPRK mutant in terms of both autophosphorylation and histone phosphorylation activity and induces the same in vivo phosphorylation pattern as wild type SPRK. However, this catalytically active SPRK zipper mutant is unable to activate JNK. Our data show that the monomeric SPRK mutant fails to phosphorylate one of the two activating phosphorylation sites, Thr(258), of MKK4. These studies suggest that zipper-mediated SPRK oligomerization is not required for SPRK activation by Cdc42 but instead is critical for proper interaction and phosphorylation of a downstream target, MKK4.

Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH(2)-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.

Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase.

Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein. The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300. Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction. Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate. Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA. The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E. coli. Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2.

Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial "overshoots" were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a "two-base" mechanism accompanied by a kinetic isotope effect. "Competitive deuterium washout" experiments were used to measure kinetic isotope effects on Vmax/Km of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by "double competitive deuterium washout" experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme.

The mechanism of the cofactor-independent glutamate racemase from Lactobacillus has been studied. The possible formation of an acylenzyme intermediate during catalysis has been investigated using 18O-carboxyl labeled glutamate. The absence of any washout of label during racemization argues against intermediate formation. The observation of the enzyme-catalyzed incorporation of deuterium at the C-2 position of glutamate upon racemization in D2O provides evidence for a deprotonation/protonation mechanism. Further experiments have been performed in order to determine the number of enzymic bases responsible for racemization. Solvent deuterium is efficiently incorporated into the product enantiomer but not into the recovered substrate enantiomer in each reaction direction. This finding is consistent with a "two-base" mechanism in which one enzymic base deprotonates the substrate, and the conjugate acid of a second enzymic base protonates the resulting intermediate from the opposite face. It also suggests that the two bases are monoprotic. The possibility that the two enzymic forms, which differ at the very least by the protonation states of the active-site bases, are kinetically significant has been examined by measuring the entire time course of the approach to equilibrium at various concentrations of glutamate. An "oversaturated" regime [Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529-2537] was not observed using glutamate concentrations as high as 100 mM, indicating that the two enzyme forms are rapidly interconverting under physiological conditions.

Protected deoxynucleoside 3'-O-ethyl-N,N-diisopropylphosphoramidite reagents were prepared for use in the automated synthesis of ethyl phosphotriester (Et) modified oligonucleotides. The title diastereomers were separated by reversed-phase HPLC, and chirality at phosphorus was assigned by an improved configurational correlation scheme that was verified by NMR spectroscopic studies (accompanying paper, Part VI). This generally applicable correlation scheme involved enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; phosphite triester sulfurization to obtain diastereomeric O-ethyl phosphorothioates, d[AS(Et)T], which were separated by HPLC for stereoretentive oxidation with H2O2 to give d[A(Et)T], and stereoretentive de-ethylation with PhSH-Et3N to give diastereomeric phosphorothioates, d[AST], whose configurations at phosphorus had been assigned previously. Neither the Rp-Rp nor Sp-Sp duplex, (d[GGAA(Et)TTCC])2, was cleaved by EcoRI endonuclease under conditions that led to cleavage of both the unmodified duplex, [d(GGAATTCC)]2, and the mixture of diastereomeric phosphorothioate-modified duplexes, [d(GGAASTTCC)]2. Cleavage of the latter substrates was Sp-selective.

Ozonolysis of compounds with the general structure CH2 = CHR1CHR2CH2OP(O) (NHR3)NR42 gave, in each case, one major product which was an analogue of the cyclophosphamide (CP) metabolites aldophosphamide (AP) or 4-hydroxy-CP. 31P NMR spectra recorded for each of these compounds in aqueous buffered solutions at pH 7.4, 37 degrees C, revealed a cascade of reactions similar to those observed for AP and/or 4-hydroxy-CP, although the individual rate constants for these reactions were substituent-dependent. Aryl ketone analogues of AP did not give rise to detectable amounts of their cyclic hemiaminals, but those which produced phosphoramide mustards at rates similar to that found for AP were active against L1210 lymphoid leukaemia in mice. The results indicated that oncostatic selectivity may not require cell-specific oxidative detoxification.

High-performance liquid chromatography (HPLC) and 1H/31P nuclear magnetic resonance (NMR) spectroscopy were used to measure the molar ratio of oligodeoxyribonucleotide products in mixtures obtained with automated DNA synthesizers that employed competitive coupling of either standard methyl- or newer beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, which include deoxyinosine. Mixtures of these reagents when used as freshly prepared solutions afforded ratios of products that indicated negligibly small differences among the rates of the various competitive coupling reactions. However, studies of reagent stability in solution revealed that both types of the N-isobutyryl deoxyguanosine reagent decompose faster than their corresponding dA, dC, and dT phosphoramidites, which led to significantly lower proportions of dG-containing sequences. This problem was attenuated for the beta-cyanoethyl reagents due to their slower rate of decomposition.

Phenyl ketone phosphorodiamidates [C6H5C(O)CH2CH2OP(O)NHR1NR2R3] were synthesized in conjunction with an ongoing investigation into the effects of substituents on the dynamical solution chemistry of the metabolites of cyclophosphamide (1a). In contrast to aldophosphamide (3a), which readily interconverts with its cyclic isomer 4-hydroxycyclophosphamide (2a), phenylketophosphamide (14a: R1 = H, R2 = R3 = CH2CH2Cl) exhibited an apparent "resistance" toward an intramolecular addition reaction such that 4-hydroxy-4-phenylcyclophosphamide (13a) could not be detected either spectroscopically (31P or 13C NMR) or chemically (NaCN trapping experiment). Control studies that compared the relative reactivities of 14a and methylketophosphamide [20: CH3C(O)CH2CH2OP(O)NH2N-(CH2CH2Cl)2] revealed that the factors that modulate the ring closure/opening reactions were not peculiar to the phenyl group; however, differences between phenyl and methyl profoundly influenced the rates of fragmentation of 14a and 20. 31P NMR spectroscopy was used to determine the rates at which each compound generated a cytotoxic alkylating agent. Under a standard set of reaction conditions [1 M lutidine buffer with added Me2SO (8:2), pH 7.4, 37 degrees C], the half-lives of 2a/3a, 14a, phenylketoifosfamide (14b: R1 = R2 = CH2CH2Cl, R3 = H), phenylketotrofosfamide (14c: R1 = R2 = R3 = CH2CH2Cl), and 20 were 72, 66, 63, 56, and 173 min, respectively. Analogues 14a and 14b exhibited good anticancer activity against a variety of test systems.

Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies have been carried out with the oligodeoxyribonucleotide mismatch sequence, d(CGCGATTCGCG), 1. It has been found that 1 exists, in solution, as an equilibrium mixture of slowly interconverting, structured conformational isomers, 1a and 1b. On the basis of the concentration dependence of the 1a-1b equilibrium, the 1H NMR spectrum of the imino protons of the nucleotide bases, and the individual CD spectra of 1a and 1b, it is suggested that the two species correspond to a B-type DNA duplex and a single-stranded, hairpin-loop structure; the portion of the single-stranded species not involved in the loop appears to have a B-type DNA structure (on the basis of the CD measurements). To facilitate 1H NMR resonance assignments, the two possible des-methyl thymidine derivatives of 1 were synthesized; the effect of this substitution on the physical chemical properties of 1 was explored. The 1H NMR spectra of 1, as a function of temperature, showed that, under conditions wherein both species were present to a significant extent, the duplex form melted at a lower temperature than the single-stranded, hairpin loop structure.