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Supplementary Table 1: Description of TCGA derived mRNA expression cohorts used to examine LIF expression and associated transcript signaturesSupplementary Table 2: Transcription signatures used to identify biological processes and cell types correlated with LIF expression.Supplementary Table 3: GSEA results of REACTOME pathway gene sets for enriched gene sets in LIF treated macrophages. Supplementary Table 4: Leading edge genes selected from GSEA analysis of LIF treated macrophages with REACTOME pathway gene sets Supplementary Table 5: Summary of data collection statistics for crystal structures of MSC-1:LIF co-complex Supplementary Table 6: Summary of interactions between MSC-1 and LIF

Supplementary Figure 1. Transcriptomic analysis demonstrates an enrichment of immunological pathways in high LIF tumors. Supplementary Figure 2. LIF transcripts are associated with a panel of immunosuppressive macrophage/myeloid signatures across human tumor indications. Supplementary Figure 3. LIF transcripts are specifically associated with suppressive macrophage signatures across human tumor indications. Supplementary Figure 4. LIF transcripts tend to be highly expressed in aggressive tumors. Supplementary Figure 5. LIF drives polarization of immunosuppressive human macrophages. Supplementary Figure 6. RNAseq of LIF induced gene sets in human macrophages predict poor patient outcome and correlate with LIF across human tumor indications. Supplementary Figure 7. Structural analysis of MSC-1 reveals an overlapping epitope with the gp130 binding site and a free cysteine in HCDR3. Supplementary Figure 8. CT26 and MC38 tumors expression LIF protein and monotherapy MSC-1 is well tolerated. Supplementary Figure 9. MSC-1 and anti-PD1 combination is well tolerated and promotes tumor memory in responsive mice.

Purpose:Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target.Experimental Design:We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer.Results:LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice.Conclusions:Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

The major membrane currents responsible for sinoatrial and idioventricular rhythm-generation were studied in isolated rat heart preparations, perfused in Langendorff mode. The rates of whole isolated hearts beating with sinoatrial rhythm decreased with cesium and ivabradine, both blockers of the funny current, and were not affected by nickel, at a dose which blocks T-type calcium current. The sinoatrial rhythm was completely abolished by reduction or removal of sodium from the perfusate (interventions that inhibit calcium-extrusive mode of the sodium/calcium exchanger), or by nifedipine, an L-type calcium channel blocker. Idioventricular rhythm, however, was arrested only by reduction of sodium in the perfusate. Ivabradine reduced the idioventricular rate, nickel did not cause any change, while nifedipine in some cases increased it. The inferences made based on these observations are that INCX and ICaL are obligatory rhythm-generating currents in the sinoatrial node, while INCX is the only obligatory mechanism for an idioventricular rhythm. The funny current is not an obligatory requirement for sinoatrial as well as idioventricular rhythm-generation. However, it enhances the frequency of LCRs. Our results in the isolated whole heart are in corroboration with results from isolated cells.

Purpose:Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target.Experimental Design:We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer.Results:LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice.Conclusions:Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

Cleistanthus collinus leaf extracts are consumed for suicidal purposes in southern India. The boiled decoction is known to be more toxic than the fresh leaf juice. Although several compounds have been isolated and their toxicity tested, controversy remains as to which compounds are responsible for the high level of toxicity of C. collinus. We report herein that cleistanthoside A is the major toxin in the boiled aqueous extract of fresh leaves and causes death in rats in small doses. The toxicity of the boiled extract prepared in the manner described can be attributed entirely to cleistanthoside A. Cleistanthin A could also be isolated from the boiled extract, albeit in trace amounts. As hypotension not responding to vasoconstrictors is the cause of death in patients who have consumed the boiled extract, effects of cleistanthoside A on the determinants of blood pressure, namely, force of cardiac contraction and vascular resistance, were tested in isolated organ experiments. Cleistanthoside A has a direct vasoconstrictor effect; however, it inhibits ventricular contractility. Therefore, the notion that the shock in C. collinus poisoning is of vascular origin must be considered carefully, and the possibility of cardiogenic shock must be studied. We present the crystal structure of cleistanthin A and show the potency of fast NMR methods (NOAH4-BSCN-NUS) in the full spectral assignment of cleistanthoside A as a real-world sample of a natural product. We also compare the results of the NOAH4-BSCN-NUS NMR experiments with conventional NMR methods.

Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.

Summary Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2’s immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2., Graphical abstract, Highlights GPC2 is a MYCN transcriptionally regulated cell-surface oncoprotein on SCLCs GPC2 is enriched in the SCLC and neuroblastoma stem cell compartment The D3-GPC2 antibody binds a tumor-specific, GPC2 conformational epitope The D3-GPC2-PBD ADC evokes durable neuroblastoma and SCLC tumor regression, Raman et al. report that GPC2 is a MYCN transcriptionally regulated cell-surface oncoprotein that is robustly differentially expressed on neuroblastomas and SCLCs, with enriched expression in the tumor stem cell compartment. An ADC that targets a GPC2 conformational epitope induces sustained neuroblastoma and SCLC tumor regression without any apparent toxicity.

Summary Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins., Graphical Abstract, Highlights • CRISPR screen identifies SEMA6A and SEMA6B as receptors for P. sordellii lethal toxin TcsL • Soluble SEMA6A ectodomain protects mouse lungs from TcsL-induced edema • 3.3 Å cryo-EM structure of TcsL bound to SEMA6A reveals atomic details of the interaction • 15 mutations in the TcsL receptor-binding interface rewire receptor specificity, A lethal bacterial toxin that plays a major role in toxic shock syndrome uses the host semaphorin proteins as receptors, and structural analysis shows a small binding interface that can be mutated to switch specificity between very different host receptors.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 10

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

The signaling co-receptor heparan sulfate proteoglycan GPC2 is a MYCN-activated, differentially-expressed cell surface oncoprotein and candidate immunotherapeutic target in neuroblastoma. Here we build on GPC2’s attributes as a robust target for immune-based therapies by finding it is highly expressed on the most clinically aggressive human neuroblastomas and small cell lung cancers (SCLCs), with highly enriched expression in the tumor stem cell compartment. A GPC2 directed antibody-drug conjugate (ADC; D3-GPC2-PBD) induces robust and durable tumor regressions in a panel of neuroblastoma and SCLC preclinical models with diverse oncogenic drivers via induction of DNA damage and apoptosis, as well as potent bystander cell killing. Finally, we illustrate that this ADC binds a tumor specific, conformation dependent epitope of the core human and murine GPC2 proteins explaining its tumor specificity and therapeutic tolerability. These studies provide the preclinical data to support the clinical translation of ADCs targeting GPC2.

Clostridium sordellii lethal toxin (TcsL) is responsible for an almost invariably lethal toxic shock syndrome associated with gynecological C. sordellii infections. Here, using CRISPR/Cas9 screening, we identify semaphorins SEMA6A and SEMA6B as the cellular receptors for TcsL and demonstrate that soluble extracellular SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that the highly related C. difficile TcdB toxin uses to bind structurally unrelated Frizzled receptors. Remarkably, reciprocal mutations in this evolutionarily divergent surface are sufficient to switch receptor specificity between the toxins. Our findings establish semaphorins as physiologically relevant receptors for TcsL, and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Introduction: We previously identified glypican 2 (GPC2) as a MYCN-driven neuroblastoma oncoprotein that is robustly differentially expressed, including expression of a tumor specific isoform (Cancer Cell 2017). We exploited this differential expression by developing an antibody-drug conjugate (ADC; D3-GPC2-PBD) that showed potent efficacy in neuroblastoma patient-derived xenografts (PDXs)/xenografts (n=4) (AACR Annual Meeting 2018). Methods: To validate GPC2 expression, we performed GPC2-directed immunohistochemistry (IHC) on a high-risk neuroblastoma tumor microarray (n=64 tumors) and flow cytometry on 8 neuroblastoma PDXs. To determine the GPC2 epitope bound by this ADC, we performed X-ray crystallography and mutational studies. To define mechanisms of cytotoxicity, we quantified DNA damage and apoptosis, immunogenic cell death (ICD), and bystander cell killing. Finally, we performed a pan-cancer analysis for GPC2 expression using RNA sequencing, flow cytometry and IHC. Results: We confirmed GPC2 expression in most high-risk neuroblastomas by IHC (60/64 tumors) and all neuroblastoma PDXs by flow cytometry (8/8 tumors). Additionally, we found a bimodal GPC2 expression pattern in neuroblastoma PDXs (GPC2-Hi and GPC2-UltraHi populations). GPC2-UltraHi cells coexpressed increased levels of the stem cell markers CD133, CD338 and CD117 (e.g., GPC2-UltraHi cell CD133 mean increase mean fluorescent intensity [MFI]=34-fold of GPC2-Hi cells). Forced GPC2 overexpression in SY5Y neuroblastoma cells was sufficient to induce increased stem cell marker expression (e.g., SY5Y-GPC2 CD133 mean increase MFI=3.4-fold of native SY5Y cells). Next, structural studies revealed that the D3-GPC2-Fab binds to a conformational and tumor-specific core GPC2 epitope, findings validated by binding kinetics experiments with GPC2 mutants, and with flow cytometry and ADC susceptibility studies. ADC treatment induced upregulation of γH2AX, cleaved PARP1 and caspase 3, indicative of DNA damage and apoptosis. In addition, we showed translocation of calreticulin to the cell surface and HMGB1 release, consistent with ICD. ADC treatment of co-incubated GPC2-high/low expressing cells induced 10-62% more cytotoxicity than expected, consistent with potent bystander cell killing. Finally, we identified several other cancers that express GPC2 and initially focused on small-cell lung cancers (SCLCs). For SCLCs, we show that GPC2 is also transcriptionally activated by MYCN, expressed at ultrahigh levels on stem cells, is integral in tumor growth and dictates comparable ADC susceptibility. Similar studies in GPC2-expressing pediatric brain tumors are ongoing. Conclusions: Neuroblastomas robustly express GPC2, including ultrahigh levels in the stem cell compartment. The D3-GPC2-PBD ADC targets a conformational and tumor-specific GPC2 epitope and is potently efficacious against a diverse panel of GPC2-expressing cancers, supporting the clinical development of GPC2-directed ADCs. Citation Format: Kristopher R. Bosse, Swetha Raman, Samantha Buongervino, Maria Lane, Kristen Upton, Hong Cui, Benjamin Martinez, Daniel Martinez, Doncho V. Zhelev, Bruce Pawel, Dimiter S. Dimitrov, Jean-Philippe Julien, John M. Maris. Characterization and development of a GPC2 ADC for neuroblastoma and other cancers [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A26.

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 fromDictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

Leukemia Inhibitory Factor (LIF) is a member of the IL-6 family of cytokines and is involved in many physiological and pathological processes including the promotion of an immunosuppressive environment to support embryo implantation, down-regulation of autoimmune processes and the regulation of stem cell homeostasis and differentiation. In cancer, LIF is hypothesized to have a complex role tumor development and progression, creating an immunosuppressive tumor microenvironment as well as promoting the activity of cancer initiating cells (CICs). LIF is highly expressed in a subset of tumors across multiple tumor types, e.g. glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer, and correlates with poor prognosis. Given the pleiotropic role LIF is hypothesized to play in cancer, inhibition of LIF represents an exciting new therapeutic concept sitting at the intersection of two key therapeutic approaches in oncology: blockade of tumor evasion of the immune system and blockade of tumor growth via inhibition of CICs. We have identified and developed MSC-1, a first-in-class humanized IgG1 monoclonal antibody that is a potent and selective inhibitor of LIF. MSC-1 cross reacts with mouse and non-human primate LIF and inhibits LIF signaling by blocking the recruitment of gp130 to the LIF-LIFR-gp130 signaling complex. The efficacy of MSC-1 was evaluated in multiple mouse tumor models and the mechanism of action investigated. LIF inhibition with MSC-1 or shRNAs reduced tumor growth in multiple syngeneic tumor models (NSCLC, ovarian and colon), and clear target engagement was shown for MSC-1. Investigations into the mechanism of action identified that inhibition of LIF with MSC-1 reprogrammed the tumor microenvironment by decreasing immunosuppressive M2 macrophages and increasing the number of intratumoral NK cells and total/activated T cells. MSC-1 also decreased immunosuppressive M2 macrophages in an orthotopic GBM xenograft model and human GBM organotypic tumor slices in an ex vivo model. Similarly, immunosuppressive macrophage genes were decreased when monocytes were co-cultured with supernatants from a GBM cell line in which LIF expression had been knocked-down. Given the effects of MSC-1 on intratumoral immune cells, we hypothesized that MSC-1 could be effectively combined with checkpoint inhibitors and we are currently evaluating MSC-1/checkpoint inhibitor combination therapy. Taken together, these findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment will lead to clinical activity in multiple cancer indications. A Phase I dose-escalation and expansion study of MSC-1 is planned to initiate early 2018 in patients with advanced solid tumors that will incorporate target engagement and PD biomarkers, as well as safety and efficacy endpoints. Citation Format: Angus Sinclair, Robin Hallett, Patricia Giblin, Isabel Huber-Ruano, Judit Anido, Naimish Pandya, Kimberly Hoffman, Ada Sala, Monica Pascual, Vanessa Chigancas, Swetha Raman, Johan Fransson, Jean-Philippe Julien, Robert Wasserman, Jeanne Magram, Joan Seoane. MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF [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 1751.

The involvement of Hsp90 in progression of diseases like cancer, neurological disorders and several pathogen related conditions is well established. Hsp90, therefore, has emerged as an attractive drug target for many of these diseases. Several small molecule inhibitors of Hsp90, such as geldanamycin derivatives, that display antitumor activity, have been developed and are under clinical trials. However, none of these tested inhibitors or drugs are peptide-based compounds. Here we report the first crystal structure of a peptide bound at the ATP binding site of the N-terminal domain of Hsp90. The peptide makes several specific interactions with the binding site residues, which are comparable to those made by the nucleotide and geldanamycin. A modified peptide was designed based on these interactions. Inhibition of ATPase activity of Hsp90 was observed in the presence of the modified peptide. This study provides an alternative approach and a lead peptide molecule for the rational design of effective inhibitors of Hsp90 function.

Cytosolic heat shock protein 90 (Hsp90) has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, development, and morphological evolution. Model systems available for studying Hsp90 function either allow ease of manipulation for biochemical studies or facilitate a phenomenological study of its role in influencing phenotype. In this work, we have explored the use of the cellular slime mold Dictyostelium discoideum to examine cellular functions of Hsp90 in relation to its multicellular development. In addition to cloning, purification, biochemical characterization, and examination of its crystal structure, our studies, using a pharmacological inhibitor of Hsp90,demonstrate a role for the cytoplasmic isoform (HspD) in D. discoideum development. Inhibition of HspD function using geldanamycin (GA) resulted in delayed aggregation and arrest of D. discoideum development at the ‘mound’ stage. Crystal structure of the amino-terminal domain of HspD showed a binding pocket similar to that described for yeast Hsp90. Fluorescence spectroscopy, as well as GA-coupled beads affinity pulldown, confirmed a specific interaction between HspD and GA. The results presented here provide an important insight into the function of HspD in D. discoideum development and emphasize the potential of the cellular slime mold to serve as an effective model for studying the many roles of Hsp90 at cellular and organismal levels.