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Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

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.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-a-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.

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.

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr(641) in STAT6. Here, we expanded the scope of our initial in vitro structure–activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED(50) of 0.25 μg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

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.

We report a very mild synthesis of N-protected α-methylamines from the corresponding amino acids. Carboxyl groups of amino acids are reduced to iodomethyl groups via hydroxymethyl intermediates. Reductive deiodination to methyl groups is achieved by hydrogenation or catalytic transfer hydrogenation under alkaline conditions. Basic hydrodehalogenation is selective for the iodomethyl group over hydrogenolysis-labile protecting groups, such as benzyloxycarbonyl, benzyl ester, benzyl ether, and 9-fluorenyloxymethyl, thus allowing the conversion of virtually any protected amino acid into the corresponding N-protected α-methylamine.

Herein we review our progress on the development of phosphopeptide-based prodrugs targeting the SH2 domain of STAT3 to prevent recruitment to cytokine and growth factor receptors, activation, nuclear translocation and transcription of genes involved in cancer. We developed high affinity phosphopeptides (K I = 46-200 nM). Corresponding prodrugs inhibited constitutive and IL-6 induced Tyr705 phosphorylation at 0.5-1 μM in a variety of human cancer cell lines. They were not cytotoxic at 5 μM in vitro but they inhibited tumor growth in a human xenograft breast cancer model in mice, accompanied by reduced VEGF expression and angiogenesis.

Signal transducer and activator of transcription 3 (Stat3) transmits signals from growth factors and interleukin-6 family cytokines by binding to their receptors via its Src homology 2 (SH2) domain. This results in phosphorylation of Tyr705, dimerization, translocation to the nucleus, and regulation of transcription of downstream genes. Stat3 is constitutively activated in several human cancers and is a target for anti-cancer drug design. We have shown previously phosphorylation of Tyr705 in intact cancer cells can be inhibited with prodrugs of phosphopeptide mimics targeting the SH2 domain. In a series of prodrugs consisting of bis-pivaloyloxymethyl esters of 4′-phosphonodifluoromethyl cinnamoyl-Haic-Gln-NHBn, appending methyl group to the β-position of the cinnamate increased potency ca. twofold, which paralleled the increase in affinity of the corresponding phosphopeptide models. However, dramatic increases in potency were observed when the C-terminal C(O)NHBn of Gln-NHBn was replaced with a simple methyl group. In this communication we continue to explore the effects of structural modifications of prodrugs on their ability to inhibit Tyr705 phosphorylation. A set of 4-substituted prolines incorporated into β-methyl-4-phosphocinnamoyl-leucinyl-Xaa-4-aminopentamide model peptides exhibited affinities of 88–317 nM by fluorescence polarization (Pro IC50 = 156 nM). In corresponding prodrugs, Pro inhibited constitutive Stat3 phosphorylation at 10 μM in MDA-MB-468 breast tumor cells. However, 4,4-difluoroproline and 4,4-dimethylproline resulted in complete inhibition at 0.5 μM. These results suggest that the prodrug with native proline undergoes metabolism that those with substituted prolines do not. In conclusion, changes in structure with minimal impact on intrinsic affinity can nevertheless have profound effects on the cellular potency of prodrug inhibitors of Stat3.

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relationship studies on phosphopeptide mimics targeted to the SH2 domain of Stat3. Inclusion of a methyl group on the β-position of the pTyr mimic 4-phosphocinnamide enhanced affinity 2- to 3-fold. Bis-pivaloyloxymethyl prodrugs containing β-methylcinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent, completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5-1 μM in a variety of cancer cell lines. The inhibitors were selective for Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby showing clear distinction between cytotoxicity and effects downstream of activated Stat3.

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.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcriptional factors that play an important role in cytokine and growth factor signaling. Here we report a 3.05 A-resolution crystal structure of an unphosphorylated STAT3 core fragment. The overall monomeric structure is very similar to that of the phosphorylated STAT3 core fragment. However, the dimer interface observed in the unphosphorylated STAT1 core fragment structure is absent in the STAT3 structure. Solution studies further demonstrate that the core fragment of STAT3 is primarily monomeric. Mutations corresponding to those in STAT1, which lead to disruption of the core fragment interface and prolonged tyrosine phosphorylation, show little or no effect on the tyrosine phosphorylation kinetics of STAT3. These results highlight the structural and biochemical differences between STAT3 and STAT1, and suggest different regulation mechanisms of these two proteins.

Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.

Both presented basic hydrodehalogenation methods are selective for the iodomethyl group over hydrogenolysis-labile protecting groups.

Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (β2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related β2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related β2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.

Under acidic conditions, the N(1)–C(4) bond of 4-(4′-hydroxyphenyl)-azetidine-2-ones are cleaved with the formation of a stabilized benzylic carbocation intermediates. The intermediates were reduced by silanes or participated in intramolecular or intermolecular Friedel–Crafts reactions to produce tyrosine mimetics.

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.

Backgrounds & Aims: Bile acids differentially modulate hepatocyte injury in cholestasis. Although glycochenodeoxycholate (GCDC) induces Fas-mediated hepatocyte apoptosis, taurochenodeoxycholate (TCDC) simultaneously activates a phosphatidylinositol 3-kinase (PI 3-K)-mediated survival pathway blocking Fas apoptosis. In this study, the mechanisms by which the TCDC/PI 3-K survival signal disrupts Fas signaling were examined. Methods: Studies were performed in primary cultures of mouse hepatocytes and the bile-salt–transporting McNtcp.24 rat hepatoma cell line. Results: GCDC, but not TCDC, resulted in cytochrome c release demonstrating that TCDC blocked apoptosis upstream of mitochondria. In contrast, both GCDC and TCDC treatment resulted in Fas aggregation and recruitment of a dominant-negative FADD green fluorescent protein (GFP) and C360S procaspase 8-GFP to the plasma membrane. Despite recruitment of procaspase 8 to the plasma membrane by both bile acids, only GCDC resulted in increases of caspase 8 activity and Bid-GFP mitochondrial translocation. However, when PI-3K was inhibited with wortmannin or dominant-negative PI 3-K, TCDC-induced Bid-GFP mitochondrial translocation and cytochrome c release. Conclusions: The TCDC/PI 3-K survival signal blocks Fas-mediated apoptosis by preventing caspase 8 activation and Bid mitochondrial translocation. Potentiation of this survival pathway in cholestasis has the potential to attenuate liver injury. GASTROENTEROLOGY 2001;120:1810-1817

In situ generation of molecular hydrogen by addition of triethylsilane to palladium-charcoal catalyst results in rapid and efficient reduction of multiple bonds, azides, imines, and nitro groups, as well as benzyl group and allyl group deprotection under mild, neutral conditions.

O-Carbamoylserine and O-carbamoylthreonine are glutamine analogues that were incorporated into a Stat3 inhibitory peptide to probe the requirements of Gln at the pY+3 position. Fmoc-Ser-NHBn and Fmoc-Thr-NHBn were converted to nitrophenyl carbonates and were attached to Rink resin via a side-chain carbamate linkage. After assembly of the peptide, acid treatment resulted in O-carbamoylserine and O-carbamoylthreonine-containing peptides. The order of affinity for Stat3 was Gln > Ser(CONH2) ≫ Thr(CONH2) suggesting a relatively tight binding pocket for the side chain of glutamine.

An improved synthesis of a phosphopeptidomimetic prodrug targeting the Src Homology 2 (SH2) domain of signal transducer and activator of transcription 6 (STAT6) is reported. In our convergent methodology, we employed a phosphotyrosine surrogate active ester harboring pivaloyloxymethyl groups, which efficiently coupled to tert-butylglycinyl proline diarylamide. Biological evaluation of 1 has not been reported. We show that it inhibits STAT6 phosphorylation in intact human bronchial epithelial cells, suggesting potential application in the treatment of asthma.

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.

An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using copper catalysed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis to an aldehyde, and condensation with sulphanilamide. This method provided multi-gram access of aprocoxib in good yield. Apricoxib exhibited potency equal to celecoxib at inhibition of prostaglandin E2 synthesis in two inflammatory breast cancer cell lines.

In cancer cells, signal transducer and activator of transcription 3 (Stat3) participates in aberrant growth, survival, angiogenesis, and invasion signals and is a validated target for anticancer drug design. We are targeting its SH2 domain to prevent docking to cytokine and growth factor receptors and subsequent signaling. One of the important elements of the recognition sequence, pTyr-Xxx-Xxx-Gln, is glutamine. We incorporated novel Gln mimics into a lead peptide, pCinn-Leu-Pro-Gln-NHBn, and found that a linear, unconstrained side chain and carboxamide are necessary for high affinity, and the benzamide can be eliminated. Replacement of Gln-NHBn with (R)-4-aminopentanamide or 2-aminoethylurea produced inhibitors with equal or greater potency than that of the lead, as judged by fluorescence polarization (IC(50) values were 110 and 130 nM, respectively). When Pro was replaced with cis-3,4-methanoproline, the glutamine mimic, (4R,5S)-4-amino-5-benzyloxyhexanamide resulted in an IC(50) of 69 nM, the highest affinity Stat3 inhibitor reported to date.

The synthesis of prodrugs targeted to the SH2 domain of Stat3 is reported. Using a convergent strategy, the pivaloyloxymethyl phosphonodiester of pentachlorophenyl 4-phosphonodifluoromethylcinnamate, a phosphotyrosine surrogate, was synthesized and used to acylate peptidomimetic fragments that were prepared on solid supports. Two prodrugs described here inhibited the phosphorylation of Stat3 in breast tumor cells.

Signal transducer and activator of transcription 3 (Stat3) is involved in aberrant growth and survival signals in malignant tumor cells and is a validated target for anticancer drug design. We are targeting its SH2 domain to prevent docking to cytokine and growth factor receptors and subsequent signaling. The amino acids of our lead phosphopeptide, Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH(2), were replaced with conformationally constrained mimics. Structure-affinity studies led to the peptidomimetic, pCinn-Haic-Gln-NHBn (21), which had an IC(50) of 162 nM (fluorescence polarization), compared to 290 nM for the lead phosphopeptide (pCinn = 4-phosphoryloxycinnamate, Haic = (2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-4-oxo-azepino[3,2,1-hi]indole-2-carboxylic acid). pCinn-Haic-Gln-OH was docked to the SH2 domain (AUTODOCK), and the two highest populated clusters were subjected to molecular dynamics simulations. Both converged to a common peptide conformation. The complex exhibits unique hydrogen bonding between Haic and Gln and Stat3 as well as hydrophobic interactions between the protein and pCinn and Haic.

In situ generation of molecular hydrogen by addition of triethylsilane to palladium-charcoal catalyst results in rapid and efficient reduction of multiple bonds, azides, imines, and nitro groups, as well as benzyl group and allyl group deprotection under mild, neutral conditions.

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

[reaction: see text] Azabicyclo[X.Y.0]alkane amino acids are rigid dipeptide mimetics that are useful tools for structure-activity studies in peptide-based drug discovery. Herein, we report an efficient synthesis of three diastereomers of 9-tert-butoxycarbonyl-2-oxo-3-(N-tert-butoxycarbonylamino)-1-azabicyclo[4.3.0]nonane (3S,6S,9S, 3S,6R,9R, and 3S,6R,9S). Methyl N-Boc-pyroglutamate is cleaved with vinylmagnesium bromide to produce an acyclic gamma-vinyl ketone. Michael addition of N-diphenylmethyleneglycine tert-butyl ester produces the N-Boc-delta-oxo-alpha,omega-diaminoazelate intermediate, which, on hydrogenloysis, gives the fused ring system. Acidolytic deprotection followed by Fmoc-protection provided building blocks suitable for solid-phase synthesis.

Signal transducer and activator of transcription 3 (Stat3) is a cytosolic transcription factor that relates signals from the cell membrane directly to the nucleus where it, in complex with other proteins, initiates the transcription of antiapoptotic and cell cycling genes, e.g., Bcl-x(L) and cyclin D1. In normal cells Stat3 transduces signals from cytokines such as IL-6 and growth factors such as the epidermal growth factor. Stat3 is constitutively activated in a number of human tumors. Antisense and dominant negative gene delivery result in apoptosis and reduced cell growth, thus this protein is an attractive target for anticancer drug design. As part of our research on the design of Src homology 2 (SH2) directed peptidomimetic inhibitors of Stat3, in this paper we describe structure-activity relationship studies that provide information on the nature of peptide-protein interactions of a high-affinity phosphopeptide inhibitor of Stat3 dimerization and DNA binding, Ac-Tyr(PO3H2)-Leu-Pro-Gln-Thr-Val-NH2, peptide 1. There is a hydrophobic surface on the SH2 domain that can accommodate lipophilic groups on the N-terminus. Of the amino acids tested, leucine provided the highest affinity at pY+1 and its main chain NH is involved with a hydrogen bond with Stat3, presumably Ser636. cis-3,4-Methanoproline is optimal as a backbone constraint at pY+2. The side chain amide protons of Gln are required for high-affinity interactions. The C-terminal dipeptide, Thr-Val, can be replaced with groups ranging in size from methyl to benzyl. We synthesized a phosphopeptide incorporating groups that provided increases in affinity at each position. Thus, hydrocinnamoyl-Tyr(PO3H2)-Leu-cis-3,4-methanoPro-Gln-NHBn, 50, was the highest affinity peptide, exhibiting an IC50 of 125 nM versus 290 nM for peptide 1 in a fluorescence polarization assay.

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.

Because signal transducer and activator of transcription (STAT) 3 is constitutively phosphorylated in several malignant disorders and plays a role in the pathophysiology of lymphoid malignancies we sought to investigate the activity of STAT3 in CLL. Using Western immunoblotting we studied peripheral blood (PB) cells from 40 patients with CLL and found that in all patients, regardless of disease stage or PB cell count, STAT3 is constitutively phosphorylated (p) on serine 727, but not on tyrosine, residues. Because it is thought that phosphorylation of tyrosine residues is requires for activation of STAT3 we asked whether the serine phosphoryalted form of STAT3 is biologically active in CLL. First we explored whether serine pSTAT3 translocates to the nucleus. Using confocal microscopy and Western blot analysis of cytoplasmic and nuclear cellular fractions we demonstrated that serine pSTAT3 is present both in the cytoplasm and nucleus of CLL cells. Then we asked whether serine pSTAT3 binds to DNA. Using the electrophoretic mobility shift assay (EMSA) we identified both a ‘shift’ and a ‘super-shift’ with anti-STAT3 and anti-serine 727 pSTAT3 antibodies, indicating that serine pSTAT3 binds to DNA. To further elucidate the role of serine pSTAT3 in CLL we inhibited its activity. We used PM, an analog of the STAT3 inhibitor Ac-Tyr(PO3H2)-Leu-Pro-Gly-Thr-Val-NH2 (Coleman DR et al., J Med Chem48:6661, 2005). Previous studies demonstrated that PM binds to the SH2 domain of STAT3 (but not to that of STAT5) and specifically inhibits STAT3 activity. Therefore, we incubated CLL cells with increasing concentrations of PM and found that PM inhibited the phosphorylation of STAT3 on serine 727 residues and downregulated Bcl-2 protein levels in a dose-dependent fashion. Because the Bcl-2 gene is activated by STAT3 it is conceivable that serine pSTAT3 activates its transcription in CLL cells. Furthermore, PM suppressed CLL colony-forming cell proliferation and induced apoptosis of CLL cells as determined by Annexin V, suggesting that constitutive serine phosphorylation of STAT3 induces both proliferative and antiapoptotic effects in CLL cells. Taken together, our data suggest that STAT3 is constitutively phosphorylated on serine 727 residues and is biologically active in CLL. Therefore, the activity of STAT3 inhibitors should be investigated in this disease.