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Faculty of Medicine | Lund University

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HAMLET, a new concept for cancer therapy

HAMLET: STRUCTURE, MECHANISM of ACTION and THERAPEUTIC EFFICACY

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Main project goals

  • To solve the structure of HAMLET and define how different parts of the molecule approach and kill tumor cells.
  • To understand the mechanisms of tumor cell death and the difference in HAMLET sensitivity between tumor cells and healthy cells.
  • To develop HAMLET into a potent cancer therapeutic agent.

Background

Few molecules destroy cancer cells without harming healthy tissues. Instead, the side effects of current cancer drugs have become an additional therapeutic area. As a result of new technologies and conceptual approaches, more targeted cancer therapies are starting to appear but the lack of specificity for tumor cells remains a significant problem. Identifying how tumor cells differ from healthy cells is therefore a great challenge, especially mechanisms of more tumor specific cell death as a basis for more tumor selective therapies.

New concepts and innovative approaches are needed to achieve tumor specific cell death and to develop more tumor selective therapies. Increasingly, biotherapies are showing promise as anticancer agents and are still a relatively untapped source of new molecules with novel mechanisms of action.

HAMLET (Human -lactalbumin Made LEthal to Tumor cells) is a complex of partially unfolded α-lactalbumin and oleic acid that kills tumor cells and immature cells but not fully differentiated healthy cells. 

hamlet is formed from

We discovered the activity of HAMLET, while using human milk fractions to block bacterial adherence to lung carcinoma cells. One milk fraction killed the tumor cells and the molecular complex responsible for this effect was identified as a folding variant of α-lactalbumin bound to oleic acid. HAMLET is the first member in a new class of potent tumoricidal molecules is formed by self-assembly of milk proteins and oleic acid4.

saxs structure of hamlet

The human variant HAMLET kills a wide range of tumor cells in vitro and shows broad but selective therapeutic efficacy in patients and cancer models. Its efficacy as a selective killer of tumor cells has been documented in vitro and in vivo in several animal models, including human brain tumor xenografts in nude rats5, murine bladder cancer6 and colon cancer in the APCMin+/- mice7. In clinical studies, purified HAMLET from human breast milk has shown therapeutic efficacy against skin papillomas8 and dramatic effects in bladder cancer patients.

Since the discovery of HAMLET in our laboratory, we have characterized the structure of the HAMLET complex, its cellular targets and therapeutic efficacy in animal models and clinical studies.

AIM 1. STRUCTURE

The HAMLET complex is formed after partial unfolding of human α-lactalbumin by binding of 4-8 oleate residues, creating a stable protein-lipid complex4. The conformational change is driven by release of the strongly bound Ca2+ ion resulting in a loss of tertiary structure definition and increased flexibility. We have recently solved a low-resolution solution structure of HAMLET and have mapped epitopes that contribute to the tumouricidal activity.

AIM 2. MECHANISM OF ACTION

Early in vitro experiments showed that HAMLET has broad anti-tumor activity with a high degree of tumor selectivity. Arguably, such conserved features may either represent general features of tumor cells that also render them susceptible to HAMLET or may reflect the presence of specific, conserved targets critical for the cell death response. To identify properties that render tumor cells susceptible to HAMLET, we have used a combination of advanced technologies to identify targets for HAMLET, responses in tumor cells and differences between tumor cells and healthy, differentiated cells.

Sensitivity to HAMLET has been demonstrated in > 40 tumor cell lines in vitro, regardless of cell type, species and tissue origin11. Sensitivity is oncogene driven, as shown by a combination of small hairpin RNA, proteomic and metabolomics technology (with Cold Spring Harbor and Berkeley Labs). Ras and cMYC are examples HAMLET sensitivity genes in tumor cells.

The death response to HAMLET is initiated by membrane perturbations, followed by inhibition of nucleotide-binding proteins (ATPases, kinases and GTPases).

RECENT PAPERS

2. 1. Receptor-independent plasma membrane remodeling by HAMLET; a tumoricidal protein-lipid complex by Aftab Nadeem, James Ho C.S. Jeremy Sanborn, Douglas L. Gettel, Viviane N. Ngassam, Anna Rydström, Thomas Kjær Klausen, Stine Falsig Pedersen, Atul N. Parikh and Catharina Svanborg. SCIENTIFIC REPORTS 

Objective

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses.  Implicit in this view is that the membranes within which the receptors reside play a secondary role. This ‘’protein-centric’’ view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction.  Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells.

Design

Using a combination of artificial membrane models and tumor cells, we characterize the membrane response to HAMLET in great detail. Membrane properties are followed in real time and characterized physically, as well as functionally. Finally, Healthy cell membranes from healthy cells are shown not to undergo such changes.

Results

HAMLET induces receptor independent changes in curvature and tubulation in (A) Artificial vesicles (B) Tumor cells. (C) Healthy differentiated cells do not show membrane tubulation.
HAMLET induces receptor independent changes in curvature and tubulation in (A) Artificial vesicles (B) Tumor cells. (C) Healthy differentiated cells do not show membrane tubulation.

We present evidence that HAMLET transforms the vesicular motif in model membranes into a dense tangle of tubules and grossly remodels plasma membranes of tumor cells, generating a positive membrane curvature, culminating in membrane protrusions and blebs. We also show that such membrane blebs provide a new, flexible compartment for HAMLET to access critical cellular constituents, notably several activated Ras family proteins on the cytoplasmic face of the plasma membrane and inhibit their downstream activity. Finally, we show that these responses are absent in healthy, differentiated cells, which resist the tumoricidal effects of HAMLET.

Conclusions

These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptor for initiating signal transduction cascades, ultimately leading to cell death. 

2. 2. II. Targeting of nucleotide-binding proteins by HAMLET - a conserved tumor cell death mechanism by James Ho Chin Shing, Aftab Nadeem,, Anna Rydström, Manoj Puthia and Catharina Svanborg. ONCOGENE

Objective

HAMLET kills tumor cells broadly suggesting that conserved survival pathways are perturbed. The aim of this study was to identify conserved molecular motifs targeted by HAMLET and to examine if their interaction with HAMLET might explain the ability of HAMLET to kill tumor cells of diverse origins. 

Design

Using a protein microarray, we investigate if HAMLET targets protein families involved in energy metabolism and cellular homeostasis including ATPases, kinases and small GTPases. In an in vitro kinase activity assay, we demonstrate, in that about 70 % of kinases are inhibited by HAMLET and confirm kinase inhibition in HAMLET treated cells by a phosphorylation antibody microarray. 

Results

We identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35 % of all HAMLET targets in a protein microarray comprising 8000 human proteins. 

(A) HAMLET binds kinases in all branches of the human Kinome tree (blue dots). (B) Kinase inhibition by HAMLET is mapped onto the human kinome. (C) HAMLET inhibits 69 % of all kinases tested (≥ 20% inhibition cut off) and enhanced the activity of 31 kinases (≥ 120%).
(A) HAMLET binds kinases in all branches of the human Kinome tree (blue dots). (B) Kinase inhibition by HAMLET is mapped onto the human kinome. (C) HAMLET inhibits 69 % of all kinases tested (≥ 20% inhibition cut off) and enhanced the activity of 31 kinases (≥ 120%).

Target kinases were present in all branches of the Kinome tree, including 26 Tyrosine kinases (TK), 10 Tyrosine kinase-like kinases (TKL), 13 Homologs of yeast Sterile (STE) kinases, 4 Casein kinase 1 (CK1) kinases, 15 Containing PKA, PKG, PKC family (AGC) kinases, 15 Calcium/calmodulin-dependent protein kinase (CAMK) kinases and 13 kinases from CDK, MAPK, GSK3, CLK families (CMGC). HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. 

HAMLET inhibits activity of Ras and BRAF. (A) HAMLET co-localizes with Ras. (B) Co-localizes of HAMLET with BRAF (C) Inhibition of Ras activity in response to HAMLET (D) HAMLET inhibits kinase activity of BRAF.
HAMLET inhibits activity of Ras and BRAF. (A) HAMLET co-localizes with Ras. (B) Co-localizes of HAMLET with BRAF (C) Inhibition of Ras activity in response to HAMLET (D) HAMLET inhibits kinase activity of BRAF.

Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo

Conclusions

HAMLET has shown therapeutic efficacy in several animal models of cancer and the therapeutic benefits of HAMLET have been confirmed in clinical studies. Understanding the mechanism of action is therefore essential. The present study defines HAMLET as a ligand of nucleotide binding proteins, including ATPases, kinases and GTPases and suggests that their inhibition leads to cell death. Specifically, HAMLET inhibited Ras and Braf activity, blocking pathways involved in proliferation and survival, explaining how the sensitivity to HAMLET can be determined by oncogenes like cMYC and Ras [12], previously defined in an shRNA screen. 

2. 3. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET. James Ho CS, Hendrik Sielaff, Catharina Svanborg and Gerhard Grüber. JOURNAL of MOLECULAR BIOLOGY

Objective

HAMLET (Human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels.

Results

A dose dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding and catalytic subunits α and ß of the energy converting F1FO ATP synthase was detected. As shown by fluorescence correlation spectroscopy HAMLET binds to the F1-domain of the F1FO-ATP synthase with a dissociation constant (KD) of 20.5 µM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ-complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ-complex interaction. Whereas the α3β3γ-complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8 ± 0.7 s-1 no rotation could be observed in the presence of bound HAMLET.

the molecular motor f atp synthase is targeted by the tumoricidal protein hamlet

HAMLET interacts with ATP synthase and inhibits its activity: (A) HAMLET (red) colocalizes with ATP synthase (green) in lung carcinoma cells. (B) Experimental setup for the single-molecule rotation assay of recombinant α3β3γ complex. (C) Dose-dependent decreases in the specific ATPase activity of α3β3γ after incubation with HAMLET.

Conclusions

The present study reports qualitative and quantitative studies demonstrating the direct binding between HAMLET and the F1-domain of the F-ATP synthase and functional consequences of this interaction. The HAMLET-F-ATP synthase association reduces enzymatic activity and rotary motion of the motor protein F-ATP synthase. Being the key enzyme in the process of oxidative phosphorylation, a reduction in the catalytic activity of the F-ATP synthase inhibits ATP-formation and reduces cellular ATP levels. As glycolysis, which tumor cells are heavily dependent on, is driven by ATP in the first rate-limiting step, a reduced F-ATP synthase function caused by HAMLET is likely to impair glycolysis and thereby drive energy-deprived tumor cells to their death.

Aim 3. Therapeutic efficacy of HAMLET

HAMLET is active as a therapeutic agent in animal models and human tumors. HAMLET treatment delayed the progression of human glioblastoma xenografts in nude rats and increased survival, triggering apoptotic changes in the tumor without evidence of cell death in healthy brain tissue. In a placebo-controlled clinical study, topical administration of HAMLET removed skin papillomas, without side effects and in patients with bladder cancer, local instillations of HAMLET killed tumor cells but not healthy cells in surrounding tissues. In addition, HAMLET triggered rapid shedding of tumor cells into the urine and caused a reduction in tumor size in patients with bladder cancer. A therapeutic effect of HAMLET against bladder cancer was confirmed in an animal model.

A. Xenograft model in which human GBM tumour spheroids (injected at the arrow) were allowed to establish for 1 week before a 24-h infusion with HAMLET (n = 19) or -lactalbumin (n = 10). B and C. MRI scans of individual tumors in rats treated with -lactalbumin (1-4) or HAMLET (5-8), were performed 7 weeks post infusion. D. The mean tumor size was significantly smaller in the HAMLET-infused animals than the -lactalbumin-treated group (P < 0.01). E. Symptoms of elevated intracranial pressure were recorded and occurred after about 2 months in the -lactalbumin controls, but the onset of pressure symptoms was delayed in rats receiving HAMLET (P < 0.001).

A. Xenograft model in which human GBM tumour spheroids (injected at the arrow) were allowed to establish for 1 week before a 24-h infusion with HAMLET (n = 19) or -lactalbumin (n = 10). B and C. MRI scans of individual tumors in rats treated with -lactalbumin (1-4) or HAMLET (5-8), were performed 7 weeks post infusion. D. The mean tumor size was significantly smaller in the HAMLET-infused animals than the -lactalbumin-treated group (P < 0.01). E. Symptoms of elevated intracranial pressure were recorded and occurred after about 2 months in the -lactalbumin controls, but the onset of pressure symptoms was delayed in rats receiving HAMLET (P < 0.001).

macroscopic response to treatment

Macroscopic response to treatment. Skin papillomas from three patients are shown: Panels A, D, and G, at enrollment (baseline); Panels B, E, and H, after the first three weeks of HAMLET treatment (lowest volume); and Panels C, F, and I, at follow-up approximately two years later. After the first phase of treatment, Patient 1 had complete resolution of the lesion, and Patients 2 and 3 had a resolution of 75 percent or more in lesion volume.

macroscopic changes in papillary tumors after hamlet exposure

Macroscopic changes in papillary tumors after HAMLET exposure. The tumor is shown before (left panels) and after (right panels) the 5 HAMLET instillations. Changes in tumor size in individual patients are illustrated by the drawings next to each pair of photographs. The diathermia loop is 5-mm wide. Patient numbers are below each schematic.

3. 1. Prevention and treatment of colon cancer by peroral administration of HAMLET Manoj Puthia, Petter Storm, Aftab Nadeem, Sabrina Hsiung and Catharina Svanborg. GUT 

Objective

Most colon cancers start with dysregulated Wnt/β-catenin signaling and remain a major therapeutic challenge. Based on the properties of the HAMLET (human alpha-lactalbumin made lethal to tumor cells) complex and its biological context, we investigated if HAMLET can be used for colon cancer therapy and prevention. ApcMin/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal tumors, were used as a model for human disease.

Design

HAMLET was given perorally in therapeutic and prophylactic regimes. Tumor burden and animal survival were compared between HAMLET treated and sham fed mice. Tissue analysis focused on Wnt/β-catenin signaling, proliferation markers and gene expression, using, microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells.

manoj prototype

Results

Peroral HAMLET administration reduced tumor progression and mortality in ApcMin/+ mice. HAMLET accumulated specifically in tumor tissue, reduced β-catenin and related tumor markers. Gene expression analysis detected Wnt signaling inhibition and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localization through an ion channel dependent pathway, defining a novel mechanism controlling β-catenin signaling. Remarkably, tumor development was also significantly prevented, by supplying HAMLET to the drinking water when they stop breastfeeding. 

Conclusion

These data identify HAMLET as a new, peroral agent for colon cancer prevention and therapy, especially needed in individuals carrying APC mutations, where colon cancer remains a leading cause of death.

The paper was accompanied by an editorial in Nature Reviews Gastroenterology and hepathology (see the following link). 

Nature Reviews Gastroenterology and Hepatology. 10:126.2013, HAMLET takes a leading role on the colorectal cancer stage.Comment: Smith K. Therapy

You can check the Group members working on this project by clicking on this link.

Publication list:

Authors

Title

Journal

Ho, J., Nadeem, A., Rydström, A., Puthia, M., Svanborg, C.

Oncogene. 18;35(7):897-907. 2015

Nadeem, A., Sanborn, J., Gettel, DL., Ho, JCS., Rydström, A., Ngassam, VN., Klausen, TK., Pedersen, SF., Lam, M., Parikh, AN., Svanborg, C.

Nature Scientific Reports. 5:16432. doi: 10.1038/srep16432. 2015

Ho, JCS., Sielaff, H., Nadeem, A., Svanborg, C., Gruber G.

Journal of Molecular Biology. doi: 10.1016/j.jmb.2015.01.024. 2015.

Puthia, M., Storm, P., Nadeem, A., Hsiung, S., Svanborg, C.

Gut. 63(1): 131-142. 2013.

Ho Cs J, Storm P, Rydstrom A, Bowen B, Alsin F, Sullivan L, Ambite I, Mok KH, Northen T,Svanborg C.J

Biol Chem. 2013 Jun 14;288(24):17460-71.

Storm, P., Kjaer Klausen, T., Trulsson, M., Ho, CS., Dosnon, JM., Westergren, T., Chao, YX., Rydstrom, A., Yang, H., Pedersen, SF., Svanborg, C. A

PLoS ONE 8: e58578. 2013.

Ho, JCS., Rydstrom, A., Manimekalai, MSS., Svanborg, C., Grüber, G.

PLoS ONE 7: e53051. 2012.

Wegmann F, Gartlan KH, Harandi AM, Brinckmann SA, Coccia M, Hillson WR, Kok WL, Cole S, Ho LP, Lambe T, Puthia M, Svanborg C, Scherer EM, Krashias G,Williams A, Blattman JN, Greenberg PD, Flavell RA, Moghaddam AE, Sheppard NC, Sattentau QJ.

Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens.

Nat Biotechnol. 2012 Aug 26.

Storm P, Aits S, Puthia MK, Urbano A, Northen T, Powers S, Bowen B, Chao Y, Reindl W, Lee DY, Sullivan NL, Zhang J, Trulsson M, Yang H, Watson JD, Svanborg C.

Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis.

Oncogene. 2011 Jun 6.

Hakansson AP, Roche-Hakansson H, Mossberg AK, Svanborg C.

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

PLoS One. 2011 Mar 10;6(3):e17717.

Trulsson M, Yu H, Gisselsson L, Chao Y, Urbano A, Aits S, Mossberg AK, Svanborg C.

HAMLET binding to α-actinin facilitates tumor cell detachment. 

PLoS One. 2011 Mar 8;6(3):e17179.

Mossberg AK, Hun Mok K, Morozova-Roche LA, Svanborg C.

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes.

FEBS J. 2010 Nov;277(22):4614-25. doi: 10.1111/j.1742-4658.2010.07890.x. Review.

Mossberg AK, Puchades M, Halskau Ø, Baumann A, Lanekoff I, Chao Y, Martinez A, Svanborg C, Karlsson R.

HAMLET interacts with lipid membranes and perturbs their structure and integrity.

PLoS One. 2010 Feb 23;5(2):e9384

Mossberg AK, Hou Y, Svensson M, Holmqvist B, Svanborg C.

HAMLET treatment delays bladder cancer development.

J Urol. 2010 Apr;183(4):1590-7. Epub 2010 Feb 21.

Pettersson-Kastberg J, Mossberg AK, Trulsson M, Yong YJ, Min S, Lim Y, O'Brien JE, Svanborg C, Mok KH.

alpha-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding.

J Mol Biol. 2009 Dec 18;394(5):994-1010. Epub 2009 Sep 18.

Gustafsson L, Aits S, Onnerfjord P, Trulsson M, Storm P, Svanborg C

Changes in proteasome structure and function caused by HAMLET in tumor cells.

PLoS One. 2009;4(4):e5229. Epub 2009 Apr 14

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