Girish Daginakatte

Spatiotemporal Differences in CXCL12 Expression and Cyclic AMP Underlie the Unique Pattern of Optic Glioma Growth in Neurofibromatosis Type 1

Astrocytoma (glioma) formation in neurofibromatosis type 1 (NF1) occurs preferentially along the optic pathway during the first decade of life. The molecular basis for this unique pattern of gliomagenesis is unknown. Previous studies in mouse Nf1 optic glioma models suggest that this patterning results from cooperative effects of Nf1 loss in glial cells and the action of factors derived from the surrounding Nf1+/- brain. Because CXCL12 is a stroma-derived growth factor for malignant brain tumors, we tested the hypothesis that CXCL12 functions in concert with Nf1 loss to facilitate NF1-associated glioma growth. Whereas CXCL12 promoted cell death in wild-type astrocytes, it increased Nf1-/- astrocyte survival. This increase in Nf1-/- astrocyte survival in response to CXCL12 was due to sustained suppression of intracellular cyclic AMP (cAMP) levels. Moreover, the ability of CXCL12 to suppress cAMP and increase Nf1-/- astrocyte survival was a consequence of mitogen-activated protein/extracellular signal-regulated kinase kinase-dependent inhibition of CXCL12 receptor (CXCR4) desensitization. In support of an instructive role for CXCL12 in facilitating optic glioma growth, we also show that CXCL12 expression along the optic pathway is higher in infant children and young mice and is associated with low levels of cAMP. CXCL12 expression declines in multiple brain regions with increasing age, correlating with the age-dependent decline in glioma growth in children with NF1. Collectively, these studies provide a mechanism for the unique pattern of NF1-associated glioma growth.

Increased c-Jun-NH2-Kinase Signaling in Neurofibromatosis-1 Heterozygous Microglia Drives Microglia Activation and Promotes Optic Glioma Proliferation

Neurofibromatosis-1 (NF1) is a common tumor predisposition syndrome in which affected individuals develop benign and malignant tumors. Previous studies from our laboratory and others have shown that benign tumor formation in Nf1 genetically engineered mice (GEM) requires a permissive tumor microenvironment. In the central nervous system, Nf1 loss in glia is insufficient for glioma formation unless coupled with Nf1 heterozygosity in the brain. Our subsequent studies identified Nf1+/- microglia as a critical cellular determinant of optic glioma growth in Nf1 GEM. Using NF1 as an experimental paradigm to further characterize the role of microglia in glioma growth, we first examined the properties of Nf1+/- microglia in vitro and in vivo. Nf1+/- microglia exhibit increased proliferation and motility and express elevated levels of genes associated with microglia activation. We further show that Nf1+/- microglia harbor high levels of activated c-Jun-NH(2)-kinase (JNK) without any significant changes in Akt, mitogen-activated protein kinase (MAPK), or p38-MAPK activity. In contrast, Nf1-/- astrocytes do not exhibit increased JNK activation. SP600125 inhibition of JNK activity in Nf1+/- microglia results in amelioration of the increased proliferation and motility phenotypes and reduces the levels of expression of activated microglia-associated transcripts. Moreover, SP600125 treatment of Nf1 optic glioma-bearing GEM results in reduced optic glioma proliferation in vivo. Collectively, these findings suggest that Nf1+/- microglia represent a good model system to study the role of specialized microglia in brain tumorigenesis and identify a unique Nf1 deregulated pathway for therapeutic studies aimed at abrogating microenvironmental signals that promote brain tumor growth.

Expression profiling identifies a molecular signature of reactive astrocytes stimulated by cyclic AMP or proinflammatory cytokines

Specialized glia, termed reactive astrocytes, accompany numerous pathologic conditions affecting the central nervous system, including stroke, multiple sclerosis, and neoplasia. To better define this important cell type, we employed high-density microarray gene expression profiling using two in vitro models of reactive gliosis (stimulation with dbcAMP or IL-1beta/IFNgamma). We identified 44 differentially expressed transcripts common to both in vitro models and demonstrated that a subset of these genes are also differentially expressed in response to experimental autoimmune encephalomyelitis and focal cerebral ischemia in vivo. Moreover, this pattern of differential gene expression is not observed in hyperproliferating or neoplastic glia.

Abstract 1650: Targeting CD47- SIRPα interaction by novel peptide-based antagonists

Background: Cluster of differentiation (CD47) is a trans-membrane glycosylated protein which is upregulated in several cancers. Increased expression of CD47 on tumor cells is associated with immune evasion and cancer progression. CD47 through its interaction with signal regulatory protein alpha (SIRPα), a cell-surface molecule on macrophages inhibits phagocytosis of tumor cells. Disrupting CD47-SIRPα interactions by monoclonal antibodies targeting CD47 and recombinant SIRPα proteins have been used as therapeutic strategies for treating cancer. Our objective was to discover and develop peptide/peptidomimetic based CD47 antagonists for disrupting CD47-SIRPα interactions. Methods: Through rational design based on crystal structure of CD47/SIRPα interacting interface, we designed peptides having potential to disrupt CD47-SIRPα interactions. FACS based cellular binding assay was developed to assess the binding affinity of CD47 antagonists. SIRPα protein labelled with fluorescent dye was incubated with Jurkat T cells expressing high levels of CD47 in the presence/absence of peptides. Binding affinity was measured by decrease in fluorescence. Functional activity of the peptides was evaluated in a FACS-based phagocytosis assays, in which tumor cells were incubated with human/mouse macrophages in the presence/absence of CD47 antagonists. Results: We identified CD47 antagonists demonstrating disruption of CD47-SIRPα interaction in a cellular binding assay. These peptides significantly inhibited phagocytosis of different tumor cells by macrophages. The lead CD47 antagonist displaying good ADME properties including moderate oral bioavailability was evaluated in a B16F10 syngeneic mouse tumor model. The lead CD47 antagonist inhibited primary tumor growth as well tumor metastasis to lungs. Biomarker characterization and efficacy studies in additional tumor models are ongoing. Citation Format: Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Nagaraj M. Gowda, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Rashmi Nair, Wesley Roy Balasubramanian, Anirudha Lakshminarasimhan, Samiulla S. Dodheri, Kiran Aithal, Raghuveer K. Ramachandra, Girish Daginakatte, Murali Ramachandra. Targeting CD47- SIRPα interaction by novel peptide-based antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2017-1650

Abstract 3070: Potent and selective inhibition of CDK7 by novel covalent inhibitors

Background: Phosphorylation of the RNA polymerase II (RNAPII) in C-terminal domain (CTD) by Cyclin-dependent kinase 7 (CDK7) is an important step in cellular transcription process. Hence pharmacological modulation of CDK7 kinase activity is considered as an interesting approach to treat cancers that critically dependent on transcription to maintain their oncogenic state. Experimental procedures: Multiple series of novel covalent CDK7 inhibitors were identified by SBDD approach based on the binding mode of known CDK7 inhibitors to find early hits. Iterative medicinal chemistry efforts were performed to identify several lead compounds by optimizing the initial hits to achieve good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile. Summary: Highly potent ATP competitive covalent inhibitors of CDK7 from two distinct chemical series were identified. They show time-dependent inhibition of CDK7 enzyme activity as a proof of covalent binding and exhibit potent anti-proliferative activity in cell lines derived from various tumor types. CDK7 modulation by these compounds was also confirmed by monitoring cellular pS5RNAPII levels. Representative compounds from each series showed very good selectivity profile in broad kinase (332) panel. Lead molecules were identified based on excellent drug-like properties (solubility, permeability and good oral bioavailability). Tolerability and efficacy studies in rodent xenograft models are ongoing with selected leads to test their impact on tumor growth inhibition and to determine therapeutic window by oral administration. Conclusion: We have identified novel and selective CDK7 covalent inhibitors from two distinct chemical series with optimized drug-like properties including oral bioavailability. These compounds are being evaluated for anti-tumor activity in mouse xenograft models. Citation Format: Leena Khare Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Manoj Kumar Pothuganti, Shilpa S. Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu B. Dabbeeru, Nagaraju A, Mahankali B, Thomas Antony, Chetan Pandit, Shekar Chelur, Girish Daginakatte, Susanta Samajdar, Murali Ramachandra. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3070.

Abstract LB-317: Identification of a novel preclinical candidate for CDK7 inhibition

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have now identified a pre-clinical candidate AU-BGB-002 which is highly potent in inhibiting CDK7 in biochemical as well as cellular assays while fully efficiently engaging the target. In a panel of kinases, AU-BGB-002 shows selectivity for CDK7. A panel of cell lines derived from a diverse set of indications are sensitive to AU-BGB-002. AU-BGB-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. When tested in a xenograft model, AU-BGB-002 treatment resulted in dose dependent tumor growth inhibition in AML xenograft model with tumor stasis at a dose of 10 mg/kg. Potent inhibiton of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII RNAPolII Ser5 phosphorylation in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK and toxicity studies are ongoing for this compound. In summary, we have identified a novel and selective CDK7 covalent inhibitor candidate with desirable drug-like properties that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-BGB-002 for the treatment of cancer. Citation Format: Leena K. Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Aravind Basavaraju, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Shilpa Nayak, Nandish C, Amith A, Ravindra MV, Dabbeeru Madhu Babu, Nagaraju A, Suraj Tgore, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Girish Daginakatte, Susanta Samajdar. Identification of a novel preclinical candidate for CDK7 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2017-LB-317

Abstract 4798: Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL

Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ∼30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ∼90% of Waldenstrom9s macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene9s compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene9s lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile. Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4798.

Abstract 3646: Novel IRAK-4 inhibitors exhibit highly potent anti-proliferative activity in DLBCL cell lines with activating MYD88 L265P mutation

Interleukin-1 Receptor Associated Kinase-4 (IRAK-4) is a serine/threonine protein kinase belonging to tyrosine like kinase (TLK) family. IRAK-4 is one of the important signalling components downstream of IL-1/Toll family of receptors (IL-1R, IL-18R, IL-33R, Toll-like receptors). Recent studies have reported occurrence of oncogenic mutations in MYD88 in 30% of ABC diffuse large B cell lymphomas (ABC DLBCL) and 90% of Waldenstrom9s macroglobulinemia (WM). Most of ABC DLBCLs have a single amino acid substitution of proline for the leucine at position 265 (L265P) in the TIR domain of MYD88 protein resulting in constitutive activation of IRAK-4. Thus, IRAK4 is an attractive therapeutic target for the treatment of B-cell lymphomas with activating MYD88 L265P mutation. We have designed, synthesized and tested small molecule IRAK-4 inhibitors based on hits originating from Aurigene’ s compound library. These novel compounds were profiled for IRAK4 kinase inhibition, anti-proliferative activity, kinase selectivity, and drug-like properties. Furthermore, selected compounds were tested in a proliferation assay and pIRAK1 mechanistic assay using ABC-DLBCL cell lines with activating MYD88 L265P mutation, OCI-lLy10 and OCI-lLy3. We have identified a series of novel bicyclic heterocycles as potent inhibitors of IRAK-4. Aurigene Lead compound exhibited potent inhibitory activity for IRAK-4 with an IC50 of 3nM in biochemical assay. Aurigene Lead compound inhibited pIRAK1 levels, and proliferation of OCI-Ly3 and OCI-Ly10 cells with an IC501of 132nM and 52nM respectively. To the best of our knowledge, Aurigene Lead compound represents the most potent IRAK4 inhibitor reported for target modulation and anti-proliferative activity in DLBCL cell lines with activating MYD88 L265P mutation. Aurigene Lead compound has good oral pharmacokinetic profile in mice and has demonstrated excellent pharmacodynamic effect in an in vivo LPS induced TNF-α model with an ED50 of 3.8 mg/Kg in mice. Preliminary in vitro tox studies indicated clean safety profile. Demonstration of efficacy in OCI-lLy10 mouse tumor model is ongoing. In summary, a series of potent IRAK-4 inhibitors belonging to 3 different chemical series have been discovered and are being evaluated for treatment of B-cell lymphomas. Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Ravi Krishna Babu D, Sivapriya Marappan, Bhavesh Choudhary, Sreevalsam Gopinath, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, Murali Ramachandra. Novel IRAK-4 inhibitors exhibit highly potent anti-proliferative activity in DLBCL cell lines with activating MYD88 L265P mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3646. doi:10.1158/1538-7445.AM2015-3646