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The 16 page composite document outlines supplemental methods as well as Figures. Fig S1: a- In vivo tumor formation assay with subcutaneous injections of (2,000) Dual hi HT-29 cells show high tumor forming efficiency of Dual hi cells compared to unsorted cells in athymic NCr nude mice. b- Line graph showing tumor volume progression in NCr nude mice injected subcutaneously with varying amount of (100,000 - 2,000) of FACS-sorted Dual hi (CD133+/CXCR4+) HCT 116 colon cancer cells. The data shows that FACS-sorted CSCs are capable of inducing tumor even at a much lower number of cells. Fig S2: a- The Kaplan-Meier survival curves indicates significant survival in NCr nude mice bearing Dual hi induced HT-29 xenografts treated with 100mg/Kg (p=0.0025) or 200mg/Kg (p=0.014) of G2.2 compared to vehicle treated control. b- Scatter plot representation of flow cytometric analyses of Dual hi CSCs in HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells following labeling with anti-CD133 and anti-CXCR4 antibodies. G2.2-treated HT-29 spheroids (100 µM) and xenograft (200 mg/kg) derived cells show significant inhibition of Dual hi CSCs compared to respective vehicle controls. c- G2.2-treated HT-29 xenograft-derived cells show significant inhibition of CSC self-renewal (2{degree sign} and 3{degree sign} sphere formation) compared to vehicle controls. d- Scatter plot representation of flow cytometric analyses of Dual hi CSCs-induced xenograft derived cells following labeling with anti-CD133, anti-CXCR4, and anti-Annexin V antibodies as well as propidium iodide counterstaining. The Panel 1 shows significant increase in Annexin V+ (apoptotic) cells in G2.2 treated xenografts compared to vehicle controls. Upon further analyses of the G2.2 treated xenograft cells (Panel 2) to determine Annexin V+ cells in each of Dual hi (CSCs) and Dual lo (non-CSCs) population revealed a robust increase in apoptotic cells in Dual hi population only, suggesting that G2.2 selective targets CSC population. e- G2.2-treated primary HT-29 xenograft-derived cells 3{degree sign} colonospheres were retreated with G2.2 in vitro and spheroid growth was measure in the presence of the drug (4{degree sign} spheroids) as well as following withdrawal of the drug (5{degree sign} & 6{degree sign} spheroids) to determine growth and self-renewal respectively of HT-29 xenograft-derived spheroids following a re-challenge with the drug. The box-plot show a robust inhibition of CSC growth/self-renewal by G2.2 following in vivo exposure to the drug (4{degree sign}, 5{degree sign} and 6{degree sign} sphere formation) compared to vehicle controls. f- G2.2 treated secondary HT-29 xenografts-derived cells show significant inhibition of CSC self-renewal (2{degree sign}, 3{degree sign} and 4{degree sign} sphere formation) compared to vehicle controls. g- Western blot analyses of xenografts show robust inhibition of CSC makers (LGR5, DCLK1) and self-renewal factor (BMI1) in G2.2 treated HCT 116 xenografts compared to vehicle controls at days 42 post treatment initiation. The associated bar-graph represents relative densitometry value normalized to GAPDH (housekeeping protein). h- G2.2 treated HCT 116 xenograft-derived cells show significant inhibition of CSC self-renewal (4{degree sign} sphere formation) compared to vehicle controls.Fig S3: a- Flow cytometry analyses of xenograft-derived cells labelled with anti-CD133-APC and CXCR4-PE antibody demonstrating higher proportion of CSCs in Chemotherapy treated mice compared to vehicle treated controls.. b- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) compared to Veh. The xenografts were harvested on day 21 post-FUOX treatment initiation.. c- Scatter plot representation of flow cytometry analyses of HT-29 xenograft-derived cells to determine proportion of Dual hi (CSCs) cells shows, a robust and sustained inhibition of Dual hi cells by CG2.2 compared to CVeh at days 30 and 42 post-FUOX treatment initiation. Numbers in the yellow box represent % of total events in a given quadrant.. d- Unedited uncut western blots along with molecular weight markers. e- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) which was promptly inhibited by CG2.2 compared to both veh. and cVeh treatments. Fig S4: a- Line graph showing activation kinetics of two key mitogen-activated protein kinases, p38 and ERK1/2 (15min → 720min) following G2.2 (100 µM) treatment of HT-29 spheroids - shows early and sustained activation of p38MAPK. Whereas ERK1/2 showed brief activation followed by modest inhibition with G2.2. The dotted line represents basal activation state. b- Western-blot analyses of MAPK shows selective activation (increased levels of phosphorylated form) of p38 but modest inhibition of related MAPK members - JNK and ERK1/2 by G2.2 in HT-29 spheroids. Moreover, activation of p38 by G2.2 (but not its inactive analogs G1.4 and G4.1) was observed selective in HT-29 spheroids enriched in CSCs but not in their monolayer counterparts, suggesting selective activation of p38 MAPK in CSCs by G2.2. Numbers under the blot represent relative densitometric values normalized to vehicle treated controls. c- Immuno-precipitation (IP) with anti-pp38 followed by western blotting (WB) with isoform specific anti-p38 antibody as well as the corresponding bar graph representation of relative densitometry values shows activation of α and β isoforms, inhibition of Î' isoform, and no significant change in γ isoform upon G2.2 treatment (100 μM for 3hrs) in HT-29 spheroids. d- Western Blot analysis shows depletion of p38α in HT-29 cells transfected with p38α siRNA (KD) without any significant effect on the levels of other isoforms. Moreover, p38α KD significantly inhibited G2.2 induced upregulation of pp38. e- Western-blot analyses of CSC (CD133, LGR5, CD44, EpCAM) and self-renewal (CMYC, BMI1) makers shows significant inhibition of these markers by G2.2 (100µM) in scrambled transfected HT-29 spheroids. However, these effects of G2.2 were near-completely reversed in HT-29 spheroids transfected with p38α siRNA, suggesting a critical role of p38α in mediating anti-CSC effects of G2.2. GAPDH served as housekeeping control. The numbers under the blot represent relative densitometric values normalized to GAPDH and vehicle treated controls. f- Western blot analysis and the corresponding bar graph representation of relative expression of CSC markers (CD133, LGR5) and pp38 shows near-complete reversal G2.2's inhibition of levels of CSC markers (24 hours) and its induction of pp38 (3 hours) in the presence of p38α agf. (dominant negative). GAPDH was used as loading control. Fig S5: a- Scatter-plot of flow-cytometric analyses of Dual hi (CD133+/CXCR4+) cells in xenografts treated with vehicle, G2.2 (200 mg/kg), SB (10mg/kg) or SB followed by G2.2 (SG). The data shows that SB, a p38 inhibitor, reversed the effect of G2.2 caused inhibition of CSCs in vivo. Fig S6: a- Average weight of mice treated with 200mg/Kg of G2.2, 3 times a week for 5 weeks compared to vehicle treated controls.. b- High resolution (20X) photomicrograph of G2.2 treated liver shows minor (~ 5%) dropout of hepatocytes (shown with bold black arrows) compared to vehicle controls.

High-dose acetaminophen (AAP) with delayed rescue using n-acetylcysteine (NAC), the FDA-approved antidote to AAP overdose, has demonstrated promising antitumor efficacy in early phase clinical trials. However, the mechanism of action (MOA) of AAP's anticancer effects remains elusive. Using clinically relevant AAP concentrations, we evaluated cancer stem cell (CSC) phenotype in vitro and in vivo in lung cancer and melanoma cells with diverse driver mutations. Associated mechanisms were also studied. Our results demonstrated that AAP inhibited 3D spheroid formation, self-renewal, and expression of CSC markers when human cancer cells were grown in serum-free CSC media. Similarly, anti-CSC activity was demonstrated in vivo in xenograft models - tumor formation following in vitro treatment and ex-vivo spheroid formation following in vivo treatment. Intriguingly, NAC, used to mitigate AAP's liver toxicity, did not rescue cells from AAP's anti-CSC effects, and AAP failed to reduce glutathione levels in tumor xenograft in contrast to mice liver tissue suggesting nonglutathione-related MOA. In fact, AAP mediates its anti-CSC effect via inhibition of STAT3. AAP directly binds to STAT3 with an affinity in the low micromolar range and a high degree of specificity for STAT3 relative to STAT1. These findings have high immediate translational significance concerning advancing AAP with NAC rescue to selectively rescue hepatotoxicity while inhibiting CSCs. The novel mechanism of selective STAT3 inhibition has implications for developing rational anticancer combinations and better patient selection (predictive biomarkers) for clinical studies and developing novel selective STAT3 inhibitors using AAP's molecular scaffold.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.

Among the biophysical techniques used to study glycosaminoglycan (GAG)-protein interactions, fluorescence spectroscopy is a quantitative tool that has been extensively used to provide structural and dynamical information. Its advantages include high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. A large majority of protein-GAG systems have been studied using either intrinsic (e.g., Trp) or extrinsic (e.g., a noncovalent fluorophore) probes. It forms the basis for measurement of dissociation constant and stoichiometry of GAG-protein complexes. We describe step-by-step procedures to measure the affinity of GAG-protein complexes, parse the ionic and non-ionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs in vivo. G2.2-reduced CSCs (CD133+/CXCR4+, Dual hi) induced HT-29 and HCT 116 colon xenografts’ growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs’ abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro, in vivo, and ex vivo mechanistic studies, we showed that G2.2′s inhibition of CSC self-renewal was mediated through activation of p38α, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs.

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa's active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.

Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide 8 showed a moderate antiproliferative potency (HCT-116 cell line inhibition of 32.0 µM), no inhibition of normal cell lines C-166, and proven cellular activities leading to apoptosis. SAR studies led to more than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 45 killed colon cancer cells at IC50 = 0.89 µM (Caco-2), 2.85 µM (HCT-116) and 1.65 µM (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC50 = 71 µM and 77.6 µM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part as a result of oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of covalently acting Michael addition compounds that potently kill cancer cells by a defined mechanism, with prominent selectivity profile over non-cancerous cell lines.

Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric inhibition of thrombin against fibrinogen (ΔY = 55-75%), the first time that a small molecule (MW 800) has been found to thwart macromolecular cleavage by a monomeric protease in a controlled manner. The work leads to the promising concept that it should be possible to develop allosteric inhibitors that reduce clotting, but do not completely eliminate it, thereby avoiding major bleeding complications that beset anticoagulants today.

Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide 1512 showed a moderate antiproliferative potency (HCT-116 cell line inhibition of 32.0 µM), good selectivity profile (no inhibition of normal cell lines), and proven cellular activities leading to apoptosis. SAR studies led to more than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 4112 killed colon cancer cells at IC50 = 0.89 µM (Caco-2), 2.85 µM (HCT-116) and 1.65 µM (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC50 = 71 µM and 77.6 µM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part as a result of oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of covalently-acting Michael addition compounds that potently kill cancer cells by a defined mechanism, with minimal effect on normal noncancerous cell.

Introduction: Earlier, we discovered that heparin hexasaccharide (HS06), but not shorter or longer polymeric glycosaminoglycan (GAGs), selectively inhibit cancer stem cells (CSCs). Insulin-like growth factor-1 (IGF1) receptor (IGF1R) plays a critical role in regulating CSC phenotype. However, unlike many growth factor receptors, GAG and IGF1R interaction and consequences have not been characterized. Methods: Colon cancer HT29 and HCT-116 cells were grown on ornithine as spheroids. Stable IGF1R knockdown cells (KD) were generated using shRNA transfection and selection. Immunofluorescence (IF) studies were performed with fluorescent-tagged HS06, GAG polymer, and pIGF1R antibody. Images were obtained using a Cytation 5 and analyzed using Gen5. Computational and molecular dynamics studies were employed to determine the binding site and nature of the interaction between HS06 and IGF1R. The impact of this interaction on activation and CSC phenotype was determined using standard techniques such as western blotting and Q-PCR. Results: Fluorescent-tagged HS06 and polymeric GAG, both bound to the colon cancer cell membrane (CM) in a dose-dependent fashion. Intriguingly, HS06 but not polymeric GAG's binding to the cell surface was abolished by IGF1R knockdown, suggesting IGF1R as a specific target HS06. Computational studies identified L1 and CR domain in the N-terminus of IGF1R as a binding site for HS06 but not multimeric GAGs. Intriguingly, there is an overlap between HS06 and IGF1 binding sites on IGF1R. Pretreatment with three different IGF1R N-terminus targeting antibodies reversed HS06 binding to CM, suggesting the N-terminus domain as a likely site of HS06 and IGF1R interactions. Additionally, IGF1 pretreatment also reversed the binding of HS06 in a dose-dependent fashion. Functionally, HS06 (but not polymeric GAG) inhibited IGF1R phosphorylation and CSC phenotype (2° spheroids and/or CSC markers). Both these effects were reversed by pretreatment with IGF1 suggesting competitive inhibition of IGF1 induced activation of IGF1R by HS06. Molecular dynamics studies identified key N-terminus residues in the L1 and CR domain of the N-terminus portion of the IGF1R distinct from IGF1 binding residues. Key basic amino acid residues that were predicted to bind specifically to HS06 in MD simulation have been observed to be mutated in cancer, and IF studies in HT29 cells expressing HS06 binding-site mutant proteins are ongoing. Conclusion: We have uncovered a novel, specific and functionally relevant interaction between HS06 and the N-terminal domain of IGF1R. The HS06-IGF1R interaction induces competitive inhibition of IGF1 induced activation of IGF1R and CSC phenotype. The findings significantly impact an understanding of GAG mediated regulation of the CSC phenotype and present a highly novel mechanism of targeting IGF1R to develop novel anti-cancer agents Citation Format: Rio S. Boothello, Jyothi C. Sistla, Nehru Viji Sankaranarayanan. Identification of insulin-like growth factor 1 receptor as a novel and specific target of heparin hexasaccharide that selectively inhibits cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3088.

Heparan sulfate (HS), a glycosaminoglycans of considerable structural diversity, has been implicated in a wide-variety of complex roles including regulation of cell signaling. Unfortunately, homogeneous HS is a dream that will likely be unfulfilled this century. To combat this, our lab has synthesized an array of non-saccharide glycosaminoglycan mimetics (NSGMs) that act as structural and functional mimetics of HS. A key advantage of our NSGMs is homogeneity, which enables assessing selectivity for recognition of target proteins with a high degree of confidence. A second advantage is the quantitative measurement of the affinity of these interactions, which affords a detailed understanding of the forces governing recognition of different proteins. Previously, we have shown a lead NSGM named G2.2 as a potent and selective inhibitor of colorectal cancer stem cells (CSCs) through phenotypic in-vitro and in-vivo assays. To improve upon the drug-like properties of G2.2, cholesterol-modified analogs (G2C, G5C, G8C) were designed to enhance potency and selectivity for protein target(s). Although the hypothesis is that G2.2 and its lipidic analogs target a ligand and/or a receptor belonging to CSC-relevant growth factors, the preferred target, if any, remains to be identified. More importantly, the relative order of preference in-vitro and its use in predicted in-vivo activity remains to be determined. In this work, we have performed a comprehensive biophysical screening of a panel of potential growth factor targets using biophysical methods to determine binding affinities, followed by quantitative measurement of the relative forces of interactions involved in target recognition. Our studies are complemented by computational experiments that provide a foundation for thermodynamic studies. Analysis of our results indicate that these NSGMs preferentially recognize certain growth factor proteins, suggesting that appropriately designed HS mimetics can be tailored to modulate a particular tyrosine kinase receptor pathway. More importantly, our results convey the principle that despite the presence of multiple sulfate groups, HS mimetics can be designed to exhibit selectivity for certain growth factor proteins. In fact, the sulfate groups on our molecules may actually contribute to the selectivity and potency of recognition, two central principles for the development of drugs. Citation Format: Connor P. O'Hara, Nehru V. Sankaranarayanan, Rio S. Boothello, Bhaumik B. Patel, Umesh R. Desai. Structurally defined glycosaminoglycan mimetics exhibit preference for proteins of the growth factor family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1166.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools.

Increasing evidence supports the concept that tumor-initiating cancer stem cells (CSCs) are responsible for the relapse and reconstitution of formerly treated tumors. Previously, our lab identified a potent and selective inhibitor of colorectal CSCs, named G2.2, from a library of non-saccharide glycosaminoglycan mimetics (NSGMs). To further improve upon the potency, pharmacokinetics, and drug-like properties of G2.2, cholesterol-modified analogs were rationally designed using computational molecular modeling and molecular dynamics studies. A small group of designed NSGMs, labeled as G2C, G5C and G8C, were synthesized for biological evaluation. These agents and the parent NSGM G2.2 were studied for their ability to inhibit the growth of primary spheroids from a panel of 15 human colorectal cancer (CRC) cells representative of the consensus molecular subtypes of human CRC. Spheroid formation frequencies (SFFs) of cell lines were measured using limiting dilution assays (LDAs), and IC50 of inhibition was evaluated from dose-response curves for monolayer growth and primary spheroid growth in the presence of NSGMs. Selectivity of CSC targeting was assessed from the ratio of IC50,monolayer to IC50,spheroid. Pharmacokinetics (PK) and bioavailability in rodents were deduced following IV and PO administrations. In-vivo therapeutic potential of the NSGMs was studied in CSC-induced xenograft models aided by ex-vivo CSC phenotype characterization. Each NSGM preferentially targeted 3D spheroidal growth in comparison to monolayer cellular growth by 12- to 450-fold, suggesting excellent CSC selectivity. Cholesterol modification of G2.2 enhanced in-vitro spheroid inhibition potency across the entire panel of cell lines on average by 3- to 21-fold. G8C showed the highest inhibition potency with an IC50 of 1 to 10 μM across cell lines. Cells with higher SFF were several-fold more sensitive to NSGMs, suggesting that basal expression of CSCs could predict NSGM response. Robust reductions in tumor volumes were observed in mice treated with G5C (100 mg/kg i.p. 3x a week) following an intial-treatment with oxaliplatin and 5-fluorouracil (weekly x3) compared to vehicle controls. This reduction was concomitant with a 3.9-fold reduction in colonic crypt stemness marker LGR5+ cells and 2.6-fold decrease in xenograft-derived tertiary spheroids. No gross or organ specific toxicity was found in animals treated with G5C. Finally, cholesterol modification greatly improved PK and oral bioavailability of parent G2.2. Overall, cholesterol modification of CSC-selective G2.2 was found to enhance in vitro potency against spheroidal growth without compromising selectivity toward tumor initiating CSCs. The G2.2 analogs offered robust tumor reductions in vivo with essentially no off target toxicity, and serve as promising and novel potential cancer therapeutics. Citation Format: Connor P. O'Hara, Rio S. Boothello, Shravan Morla, Alberto Vera, Daniel K. Afosah, Nehru V. Sankaranarayanan, Balaji Nagarajan, Chetna Sharon, Bhaumik B. Patel, Umesh R. Desai. Cholesterol modification enhances potency and pharmacokinetic properties of a selective cancer stem cell targeting agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6353.

High mobility group box 1 (HMGB1) is an alarmin released from macrophages after infection or inflammation and is a biomarker of lung disease progression in patients with cystic fibrosis. We reported that 2-O, 3-O desulfated heparin (ODSH) inhibits the release of HMGB1 from murine macrophages triggered by neutrophil elastase both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this study, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase that has been linked to HMGB1 acetylation and release. ODSH inhibited both neutrophil elastase and LPS-triggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration-dependent manner. Fluorescein-labeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus, suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro, resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognizes a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (dissociation constant ∼1 nM) in a highly cooperative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.

// Nirmita J. Patel 1, 3 , Chetna Sharon 1 , Somesh Baranwal 1, 2 , Rio S. Boothello 1, 2 , Umesh R. Desai 3 , Bhaumik B. Patel 1, 2 1 Hunter Holmes McGuire VA Medical Center, Richmond, VA 23249, USA 2 Division of Hematology, Oncology, and Palliative Care, Department of Internal Medicine and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA 3 Department of Medicinal Chemistry and Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA Correspondence to: Bhaumik B. Patel, email: bhaumik.patel@vcuhealth.org Keywords: cancer stem cells, colorectal cancer, glycosaminoglycans, p38 mitogen activated protein kinase, heparan sulfate Received: June 17, 2016 Accepted: September 05, 2016 Published: September 30, 2016 ABSTRACT Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse. STATEMENT OF SIGNIFICANCE Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.

Introduction: Cancer cell dormancy and the cancer stem cell (CSC) phenotype are interlinked and play a crucial role in disease relapse. The pp38/pERK signaling ratio positively regulates cellular dormancy. We observed that heparin hexasaccharide (HS06) inhibited colon CSC self-renewal through isoform specific induction of pp38α/β, increasing the pp38/pERK ratio, and downregulation of C-terminal binding protein-2 (CtBP2) levels which is known to play a critical role in CSC growth. Hence, we hypothesized that HS06 inhibits CSCs self-renewal and prevents dormancy escape through regulation of CtBP2 in a manner dependent on p38 activation. Method: Spheroid culture of HT29 and HCT116 colon cancer cells was used for CSC self-renewal monitoring. Transient transfection with p38α, p14ARF, CtBP2 shRNA and/or expression vector with appropriate scrambled or vector controls was used to modulate cell signaling through this pathway. Standard western blotting, Q-PCR, flow cytometry, and immunoprecipitation were used to examine protein levels, their phosphorylation status and protein-protein interactions. Short term exposure of colon cancer cells (HT29) to chemotherapy (5-Fluorouracil (125μM) and oxaliplatin (5μM) (5-FUOX)) was used to study a transient dormancy-like state followed by growth escape. HT29 CSC (1x105 CD133+/CXCR4+ cells) induced xenografts were established s.c. in NCr nude mice were treated with HS06 (150mg/kg 3xwk., 3 weeks) and monitored for tumor growth, and xenografts were analyzed for CSC makers and p38-p14ARF-CtBP2 axis status. Results: HS06 inhibited CtBP2 levels but increased the expression of its negative regulator p14 ARF in vitro and in vivo in a manner dependent on p38 activation, as SB203580, a selective p38α/β inhibitor, as well as p38α shRNA, both near-completely reversed the effects of HS06 on p14ARF, CtBP2, and CSC self-renewal. This regulation of p14ARF and CtBP2 was post-transcriptional in nature. Moreover, reciprocal coimmunoprecipitation studies revealed a novel physical interaction between endogenous pp38 and p14ARF, which resulted in serine phosphorylation of p14ARF in a p38 activation-dependent manner that was critical for regulation of CtBP2 levels and CSC self-renewal. Using a chemotherapy induced cellular dormancy model, we observed that activation/upregulation of p38 and p14ARF prevented escape from dormancy in vitro, whereas increased CtBP2 had the opposite effect. Conclusion: We have discovered a novel p38-p14ARF-CtBP2 axis that plays a dual role in colon cancer CSC self-renewal and cellular dormancy. Agents targeting this pathway e.g., HS06 and CtBP2 inhibitors, represent novel therapeutic strategies to inhibit CSCs and prevent dormancy escape leading to reduced tumor relapse and/or cure. Citation Format: Rio S. Boothello, Nirmita J. Patel, Priyadarshan K. Damle, Chetna Sharon, Kranthi Kumar Chougoni, Umesh R. Desai, Steven R. Grossman, Bhaumik B. Patel. p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3460.

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcAp2S). Genetic algorithm-based computational docking and filtering suggested that GlcAp2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcAp2S and 2-N-sulfonated glucosamine residues, labeled as HS2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcAp2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-O-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences.

Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG-protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein-GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG-protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG-protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein-GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG-protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.