Ajay Rana - US grants

DESCRIPTION (provided by applicant): MLK3, also known as SPRK, is a mitogen-activated protein kinase that can activate c-jun N- terminal kinase (JNK) and induce cell death in neuronal and non-neuronal cells. A broad-spectrum inhibitor of MLK family members (CEP-1347) is under clinical trials for treating neurodegenerative disorder related diseases like Parkinson's. TNF-a and ceramide have been shown to cause cell death during neurodegenerative disorders. Interestingly, ceramide has also emerged as a biochemical mediator of cell death in response to chemo- and radio- therapies. Studies from our laboratory have revealed that the cell death and differentiation inducing ligands, TNF-a and ceramide are potent agonists of MLK3 in mammalian cells. In addition, our more recent studies (both published and unpublished) indicate that (i) ceramides can directly associate, and activate MLK3, (ii) MLK3 mediates TNF-a and ceramide-induced JNK activation, (iii) TNF-a treatment of Jurkat cells triggers TRAF2 association with MLK3, (vi) overexpression of MLK3 causes cell death, and (v) Retinoic acid induced HL-60 cell differentiation is inhibited by MLKs specific inhibitor. Based on these observations, our major goals for this grant application are to elucidate the mechanisms of MLK3 activation by ceramide and TNF-a, and to define the physiological roles of ligand-activated MLK3 in cell death and differentiation pathways. These goals will be achieved following four specific aims. In aim 1, the in vitro and in vivo mechanisms of MLK3 activation by ceramide will be elucidated. In aim 2, the mechanism of TNF-a-induced MLK3 activation, and its synergy with ceramide-induced MLK3 activation will be examined. In aim 3, the physiological roles of MLK3 in mediating cell death and differentiation pathways will be elucidated using both biochemical and genetic approaches. In aim 4, the physiological role of MLK3 in neuronal apoptosis will be examined in MPTP mouse model (i.e. PD model), using recently created MLK3 KO and wild type animals. Elucidating the mechanism(s) of ceramide and TNF-ainduced MLK3 activation, and MLK3's role in modulating cell death and differentiation pathways will offer a basis for the development of targeted therapeutic interventions for pathologic neuronal loss occurring as a result of conditions, such as neurodegeneration, trauma or ischemia. Furthermore, the results from these studies will aide in the development of novel pharmacological approaches towards increasing the efficiency and specificity of chemo- or radio- sensitization for cancer therapies.

DESCRIPTION (provided by applicant): Mixed Lineage Kinase 3 (MLK3) is a stress-activated MAP Kinase Kinase Kinase member, whose biological function is still elusive. While dissecting the signaling pathway mediated via MLK3, we observed a strong interaction between mammalian homolog of yeast Ste20 member, p21 activated kinase (Pak1) and MLK3. Initially, we speculated that Pak1 might regulate MLK3 kinase activities similar to its yeast counterparts. However, the observed results were counterintuitive to our speculation, and instead MLK3 directly phosphorylated and activated Pak1, which subsequently lead to NF-?B activation. These results were quite intriguing because Pak1 activation has been implicated in mammary gland hyperplasia, and recently it has been identified as a major oncogene in breast cancer. Our preliminary investigation revealed that Pak1 activity was directly regulated in a MLK3-dependent manner in breast cancer cell lines. We also observed that in primary human breast tumors, the MLK3 activities directly correlated with Pak1 and NF-?B activations, exclusively in ER and PR negative breast tumors and Pak1 was overexpressed in ER- PR- breast tumors. If these results are true then the available MLKs inhibitor, CEP-1347/CEP-11004 should induce cell death in ER- breast cancer cells? Indeed, treatment of ER-, but not ER+ breast cancer cell lines with CEP-1347 caused cell death. Based on our observations, we hypothesize that in ER- breast cancer cells, MLK3 activates Pak1 and its downstream NF-?B, leading to ER- tumorigenesis. Therefore targeting MLK3 or other MLKs could abrogate ER- breast tumors. To achieve our goals, we will determine that: (1) activation of Pak1 by MLK3 induces ER- breast cell tumorigenesis, (2) disruption of MLK3-Pak1 signaling cascade attenuates ER- breast cell tumorigenesis; and (3) the mechanism of MLK3, Pak1 and NF-?B activation in ER- breast cell tumorigenesis. It is expected that by defining the role of MLK3 in Pak1 and NF-?B activation and tumorigenesis, we might control/treat ER- breast cancers by using available inhibitors of this pathway. Interestingly, the specific inhibitor of MLK3 family, CEP-1347 has been used in clinical trials for treating neurodegenerative diseases. It has also been suggested that MLKs inhibitors might serve as a treatment for specific type of cancer, underscoring our unexpected novel results related to MLK3 role in ER- breast cancer. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for difficult to treat ER- breast cancer.

The amplification of Estrogen, Progesterone and Human Epidermal Growth Factor receptors (i.e. ER, PR and HER2) serve as prognostic markers in breast cancer. The prognosis of ER+ breast cancer is relatively better due to availability of several drugs that essentially block ER-signaling. However, HER2+ breast cancer has limited options and is treated either by using humanized antibodies, Trastuzumab (Herceptin) or Pertuzumab or tyrosine kinase inhibitor, Lapatinib. The initial responses to anti-HER2 therapies are favorable, however, the tumors subsequently acquire resistance and relapse to form metastatic tumors. Therefore, there is a need to identify novel target and develop new therapeutics that can overcome anti-HER2 therapies resistance and perhaps can be used as an alternative to the existing anti-HER2 agents. Interestingly, it is well recognized that HER family-mediated signaling primarily impinge on PI3K-AKT pathway and is reported to be a main source of either, de novo or acquired resistance. Therefore several inhibitors of PI3K or AKT have been in clinical trials, however the outcome of these trials are dismal due to unacceptable toxicities. Furthermore, it is also reported that ER and IGF1R are co-amplified in HER2 positive tumors, treated with anti-HER2 therapies, leading to resistance. Both of these receptors are known to activate PI3K-AKT and is considered to be initiating anti-HER2 therapies resistance. Collectively these results suggest that PI3K-AKT is one of the main nodal point of resistance in HER2+ tumors. Therefore, any therapeutic(s) that can block PI3K-AKT signaling pathway and congruently also activate the downstream pro-apoptotic molecule will be a drug of choice to treat both HER2 therapy sensitive and resistant tumors. We reported previously that a member of Mixed Lineage Kinase (MLK) family, MLK3 was inhibited by ER, IGF1R and HER2 pathways and this inhibition was mediated via direct phosphorylation of MLK3 by AKT. We also reported that ceramide, a bioactive lipid was able to activate MLK3 and was necessary for MLK3-mediated cell death. It is reported that ceramides inactivate PI3K-AKT pathway and therefore we hypothesize that delivery of ceramides into HER2-resistant and sensitive cells might cause cell death and tumor regression. Our preliminary data clearly showed that indeed ceramide nanoparticles were able to cause cell cycle arrest and cell death in Herceptin resistant cell lines and xenografts. Based on our intriguing results, we hypothesize that ceramide nanoparticles could be a better option to treat both HER2 resistance and sensitive tumors. To determine the effectiveness of ceramide nanoparticle as an alternative option to treat both resistant and sensitive HER2+ breast cancer, we will determine: 1) the effects of ceramide nanoparticles on cell lines- and xenografts-resistant to anti-HER2 therapies, 2) the molecular mechanisms of anti-tumorigenic effects of ceramide nanoparticles in HER2+ breast cancer and finally, 3) the therapeutic efficacy of ceramide nanoparticles in HER2 transgenic and PDX animal models, resistant to anti-HER2 therapies. It is expected that at the conclusion of this project we would discern the therapeutic value of ceramide nanoparticles in treating HER2 resistant and sensitive breast cancer, and perhaps other sub-types. It is important to note that it is reported that ceramides damage specifically tumor but not normal cells and therefore we expect that our results will provide an innocuous therapeutic to treat HER2+ breast cancer with minimal, or no collateral damage.