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Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We recently demonstrated that SF2312, a natural product phosphonate antibiotic, is a potent inhibitor of the glycolytic enzyme Enolase with potential utility for the collateral lethality-based treatment of Enolase-deficient glioblastoma (GBM). However, phosphonates are anionic at physiological pH, limiting cell and tissue permeability. Here, we show that addition of pivaloyloxymethyl (POM) groups to SF2312 (POMSF) dramatically increases potency, leading to inhibition of glycolysis and killing of ENO1-deleted glioma cells in the low nM range. But the utility of POMSF in vivo is dose-limited by severe hemolytic anemia. A derivative, POMHEX, shows equipotency to POMSF without inducing hemolytic anemia. POMHEX can eradicate intracranial orthotopic ENO1-deleted tumors, despite sub-optimal pharmacokinetic properties. Taken together, our data provide in vivo proof-of-principal for collateral lethality in precision oncology and showcase POMHEX as a useful molecule for the study of glycolysis in cancer metabolism.

Purpose To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. Methods and Materials The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an “antitumor” (M1) or a “protumor” (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Results Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. Conclusions These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-06.

Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

The transcriptional regulator STAT3 curbs pro-inflammatory cytokine production mediated by NF-κB signalling in innate immune cells, yet the mechanism by which this occurs has been unclear. Here we identify STAT3 as a pivotal negative regulator of Ubc13, an E2 ubiquitin-conjugating enzyme that facilitates TRAF6 K63-linked ubiquitination and NF-κB activation. Ubc13 accumulates intracellularly in the absence of STAT3. Depletion of Ubc13 in Stat3-deficient macrophages subdues excessive RANKL- or LPS-dependent gene expression, indicating that Ubc13 overexpression mediates enhanced transcriptional responses in the absence of STAT3. In RANKL-activated macrophages, STAT3 is stimulated by autocrine IL-6 and inhibits accrual of Ets-1, Set1 methyltransferase and trimethylation of histone H3 lysine 4 (H3K4me3) at the Ube2n (Ubc13) promoter. These results delineate a mechanism by which STAT3 operates as a transcriptional repressor on Ube2n, thus modulating NF-κB activity by regulation of Ubc13 abundance. Our data suggest that this pathway plays important roles in bone homeostasis and restraint of inflammation.

Class IA phosphoinositol-3-kinases (PI3Ks) play pivotal roles in cancer and inflammation and are targets for anti-cancer therapies. p85 was first described as a regulatory subunit of Class I PI3Ks which complexes with the p110 catalytic domain to form the heterodimeric enzyme. PI3Ks are recruited to cytokine and growth factor receptors via the SH2 domains of p85. While p85 represses the kinase activity of p110 subunits in the cytosol, binding to pTyr residues on receptors or adapters activates p110, resulting in the phosphorylation of the 3’-hydroxyl group of phosphatidylinositol-4’,5’-diphosphate (PIP2) to form phosphatidylinositol-3’,4’,5’-triphosphate (PIP3). Membrane-associated PIP3 subsequently recruits kinases such as PDK1 and Akt to the cell membrane. PDK1 phosphorylates and activates Akt, which propagates growth and survival signals. In biochemical assays, doubly phosphorylated peptides enhance catalytic activity of p85/p110 dimers, which led to the dogma that phosphopeptides should activate PI3K in cells. Due to the challenges of cellular entry of negatively charged phosphopeptides, blocking protein-protein interactions mediated by p85 has been largely unexplored in intact cells. We targeted the SH2 domains of p85 with phosphatase-stable, cell-permeable prodrug analogs of Y(p)VPML, a high-affinity ligand derived from Tyr751 of PDGF. To allow cell entry and to protect against phosphatase degradation, we replaced pTyr with bis-pivaloyloxymethyl esters of 4-phosphonodifluoromethylphenylalanine (F2Pmp(POM2)), which have not been reported. We synthesized Z-NMe-F2Pmp(POM2)-OPcp with the á-amino group methylated to enhance cell penetration and the carboxyl group esterified with pentachlorophenol. Z-protection was necessary for phosphonate protecting group manipulation and for stability of the Pcp ester. A series of inhibitors was prepared by coupling Z-NMe-F2Pmp(POM2)-OPcp to analogs of VPML. Hydrogenation in the presence of Ac2O gave an N-terminal acetyl prodrug. Screening for inhibition of EGF-stimulation of pAkt in serum starved MDA-MB-468 cells led to the identification of the lead inhibitor, PM-190I. In serum starved MDB-MB-231 cells, PM-190I inhibited EGF-stimulated phosphorylation of Akt with an IC50 of 5 ìM. At 30 ìM Akt phosphorylation was negligible. These results suggest that the phosphopeptide mimic blocks recruitment to p85/p110 dimers to EGFR or its substrates, thereby preventing PI3K pathway signaling. Further, in intact cells, a mono-phosphopeptide does not stimulate PI3K activity, which contradicts current dogma. In addition to its role as a regulatory subunit, non-catalytic roles of p85 include complexing Rab4 and Rab5, stabilizing PTEN, invadopodium formation, cytokinesis, and nuclear trafficking of proteins such as XBP-1 and OPNi. Phosphopeptide mimics targeted to the SH2 domain of p85 have potential applications both in blocking PI3K signaling as well as in modulating non-catalytic functions. Citation Format: Pijus K. Mandal, Yanhua Yao, Anne R. Bresnick, Jonathan M. Backer, John S. McMurray. Peptide prodrugs targeting the SH2 domains of p85 block PI3K signaling. [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 4838.

A highly efficient synthesis of the promising Cox-2 inhibitor, apricoxib (VI), in only three steps from commercially available starting material is given.

The molecular signature of inflammatory breast cancer (IBC) includes activation of target genes of the nuclear factor-kappa B (NF-κB) transcription factor. These NF-κB target genes are differentially activated in IBC tumors and primarily produce pro-inflammatory mediators such as the chemokine interleukin-8 (IL-8), the lipid mediator prostaglandin E2, the chemokine receptor CXCR4 and its ligand partner CXCL12, and the axis defined by IL-6/Janus kinases and signal tranducer and activator of transcription 3 (STAT3). While these genes are known to regulate innate immune responses, they also are critically important to survival of tumor cells and to metastatic progression. Ongoing research is defining the roles of these inflammatory mediators and associated signaling pathways in breast cancer, in general, and in IBC. Some of these studies have evaluated pharmacological and biological agents that effectively target these pro-inflammatory mediators and have led to development of new therapeutics that may effectively abrogate IBC growth and metastasis. In summary, this chapter reviews the inflammatory mediators that have been identified as part of the molecular fingerprint of IBC and describes new evidence for the potential for inhibitors of these mediators to target specific populations of cells within IBC tumors that contribute to tumor initiation and metastatic progression.

Lung cancer is the deadliest cancer in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Regrettably response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is indispensable. It is thought that the epithelial-mesenchymal transition (EMT) plays a crucial role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor of the epithelial phenotype by regulating EMT and metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through Reverse Phase Protein Array (RPPA) profiling that high levels of miR-200 in poorly metastatic lung tumor cells can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing pharmacologic approaches to inhibit STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200 and inhibition in metastatic lung cancer could provide benefit to lung cancer patients as a potential treatment option. Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B36. doi:10.1158/1538-7445.CHTME14-B36

Lung cancer is one of the deadliest cancers in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Unfortunately response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is essential. It is thought that the epithelial-mesenchymal transition (EMT) plays a critical role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor for the epithelial phenotype by regulating EMT-inducing transcription factors such as Zeb1, and subsequent metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through reverse phase protein array (RPPA) profiling that in poorly metastatic lung tumor cells, high miR-200 levels can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing siRNA approaches or pharmacological inhibition of STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200/Zeb1 axis and could provide a potential therapeutic target in lung cancer patients. Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C57.

Signal transducer and activator of transcription 3 (Stat3) is recruited, via its SH2 domain, to phosphotyrosine residues on growth factor and IL-6 family receptors at which time it is phosphorylated on Tyr705 by Janus kinases, Src, or the kinase activity of the receptor. Phosphorylation of Tyr705 (pStat3) leads to dimerization, translocation to the nucleus, and transcription of downstream genes. Stat3 is activated in several human cancers and is considered a drug target. Starting with pTyr-Leu-Pro-Gln, we have been developing phosphopeptide mimics to target the SH2 domain with the goal of blocking recruitment to receptors to uncouple Stat3 from its signaling role. We have developed cell-permeable, phosphatase-stable prodrugs that inhibit constitutive and IL-6-stimulated Tyr705 phosphorylation at concentrations of 0.5 − 1 μM in a variety of human cancer cell lines. They are selective for Stat3 over Stat1, Stat5, Src, and PI3K. At 5 μM they are not cytotoxic to a panel of human cancer cell lines. Higher concentrations lead to off-target effects and cytotoxicity. Recent reports indicate that Ser727 phosphorylated Stat3 participates in electron transfer in the mitochodria and unphosphorylated Stat3 participates with NF-κB-mediated transcription. As opposed to Stat3 knockdown techniques which cannot discriminate the multiple roles of Stat3, our prodrugs are potential tools to selectively probe Stat3 phosphorylation. Both intra-tumoral and intra-peritoneal administration of our lead prodrug, PM-73G, resulted in reduction of orthotopic breast tumor xenografts in mice. There was no evidence of apoptosis, reduction of cyclin D1 or survivin. Treated tumors exhibited significant reduction in microvessel density. Thus, selectively inhibiting Tyr705 phosphorylation is a potential antiangiogenic treatment modality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B8.

Inflammatory breast cancer (IBC) is the most aggressive and lethal of all breast cancers. There is little known about the molecular signatures of IBC with the exception that these breast cancers commonly over-express E-cadherin and Rho C GTPase and exist as tumor emboli within dermal lymphatics. In addition, IBC tumor emboli exhibit characteristics of tumor initiating cells, i.e., cancer stem cells. Another interesting characteristic of IBC tumors is their ability to undergo vasculogenic mimicry, forming tube-like structures that abrogate the need of this very aggressive tumor type to induce the development of angiogenic endothelium. In vitro under low adherence culture conditions, IBC cells readily form tumor spheroids that contain ”label retaining” cells and exhibit self-renewal. In addition, when cultured as tumor spheroids in 3 dimensions, they express high levels of the phosphorylated form of the signal transducer and activator of transcription 3 (phospho-stat3), recently shown to regulate survival of embryonic stem cells. From a panel of newly developed novel stat3-targeted peptidomimetics, we found that the lead compound, designated as PM-73G, inhibited phospho-stat3, which was associated with blockade of the robust invasion, anchorage independent growth in soft agar and vasculogenic mimicry exhibited by IBC tumor cells without a significant anti-proliferative effect. Using array-based PCR and protein analysis, we found a specific pattern of inhibition of genes/proteins within the Jak/Stat pathway by PM-73G. Results of comparative studies using both PM-73G and stat3 shRNA demonstrate a role for stat3 in maintenance of the cancer stem cell phenotype exhibited by IBC tumor spheroids. Taken together, these studies suggest that stat3 may serve both as a molecular signature of IBC and as a target for development of effective therapeutics for this lethal form of breast cancer that contain tumor cells exhibiting a cancer stem cell phenotype. (Funded by The Susan G. Komen For The Cure Promise Grant KGO 81287 (FMR), The State of Texas Rare and Aggressive Breast Cancer Fund (FMR), NIH NCI CA096652 (JSM) and Center for Targeted Therapy at The University of Texas M.D. Anderson Cancer Center and the Texas Institute for Drug and Diagnostic Development at The University of Texas, Austin. (FMR & JSM). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 653.

Signal transducer and activator of transcription 3 (Stat3) transmits signals from IL-6 family cytokines, EGF and VEGF. Stat3 is constitutively activated in lung, head and neck, breast, and prostate cancers, AML and other malignancies, providing a target for cancer drug design. To uncouple Stat3 from its roles in proliferation, survival, angiogenesis, and invasion, we are targeting its SH2 domain with phosphopeptide mimetics derived from the recognition sequence, pTyr-Leu-Pro-Gln. Molecular modeling indicated that a methyl group attached to the β-position of a pTyr mimic would enhance affinity due to hydrophobic contact. This modification increased affinity 2-3 fold in a series of peptidomimetics based on pCinn-Xxx-Yyy-Gln-NHBn (pCinn = 4-phosphorylcinnamic acid), resulting in IC50 values of 35 − 112 nM in a biochemical fluorescence polarization assay. Conversion of these to bis-pivaloyloxymethyl (POM) protected difluoromethylphosphonate prodrugs resulted in complete inhibition of the phosphorylation of Tyr705 of Stat3 at concentrations of 0.5 − 1 μM in breast, ovarian, and melanoma cells. Prodrugs were selective for Stat3 over other SH2 domain-containing proteins such as Stat1, Stat5, Src, and PI3K p85. Quantitative RT-PCR showed that prodrugs inhibited Stat3-targeted gene expression. Prodrugs were not cytotoxic to cells grown in 2D cultures on plastic dishes. However, potent inhibition of Stat3-driven processes such as vasculogenic mimicry, invasion, and anchorage independent growth in soft agar was observed. Conclusions: 1. peptidomimetic prodrugs are highly potent and selective inhibitors of Stat3; 2. cultured tumor cells do not depend on phosphorylation of Tyr705 of Stat3 for survival; 3. highly selective inhibitors of Stat3 phosphorylation are likely to be effective antitumor agents due to inhibition of invasion and angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2527.