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HAMLET, a new concept for cancer therapy

HAMLET: STRUCTURE, MECHANISM of ACTION and THERAPEUTIC EFFICACY

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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 ca

rcinoma 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.

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.

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HAMLET – A protein-lipid complex with broad tumoricidal activity

saxs structure of hamlet

1. HAMLET triggers a broad tumoricidal response with apoptosis-like features

The early collaborative studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. The morphologic changes in dying tumor cells resembled apoptosis, with nuclear condensation, loss of cytoplasm and membrane blebbing. Using the DNA fragmentation technology of the Orrenius laboratory, HAMLET was shown to trigger typical DNA laddering. Tumor cells from different tissues shared the apoptotic features but normal cells in primary culture were resistant, suggesting an unexpected degree of tumor specificity. Compared to classical apoptosis inducers like Etoposide, the morphological changes and loss of viability were more rapid and more tumor specific.

In experiments with the Orrenius laboratory, we observed swelling and depolarization of mitochondrial membranes, accompanied by the release of Cytochrome C, activation of caspase 3, caspase 9 and phosphatidylserine exposure on the outer leaflet of the plasma membrane. These findings suggested that the cells were dying by apoptosis, initiated at the level of the mitochondria. Paradoxically, tumor cells were not rescued by the pan-caspase inhibitor ZVAD, pointing to an alternative death mechanism. In later studies, we further showed that HAMLET-induced cell death is independent of the host BCL-2 and p53 genotype. Overexpression of the anti-apoptotic proteins, BCL-2 and BCL-XL did not significantly alter the cell death response and a Tet-inducible p53 expression vector had no effect. Parallel work on the effects of HAMLET on bacteria cell death have shown that such morphological changes and biochemical responses can also be seen in prokaryotes.

2. Cellular and molecular targets

2.1. Oncogenes determine the sensitivity to HAMLET

HAMLET kills a large number of different tumor cells, in vitro, suggesting that the complex targets conserved mechanisms of cell death. In collaboration with Cold Spring Harbor Laboratory, we identified classical oncogenes like MYC, RAS and HIF1α as genetic determinants of HAMLET sensitivity in an shRNA screen, suggesting that cells targeted by HAMLET fulfill generally accepted tumor criteria. Still, the mechanism of action of HAMLET is unusual, as the complex interacts with a number of molecular targets and cellular compartments. After initial membrane interactions, HAMLET is internalized by the tumor cells, interacts with lysosomes, mitochondria and proteasomes and translocates to the nuclei. Organelle-specific molecular interactions also lead to proteasome inhibition, chromatin modification, transcriptional inhibition and death (Fig. 1).

Cellular and molecular targets
Cellular and molecular targets

2.2. Membrane perturbations and ion fluxes

HAMLET integrates into the membranes of tumor cells and triggers rapid ion fluxes, which activate downstream signaling pathways involved in cell death. Three critical features of the membrane response to HAMLET have been identified. (1) HAMLET interacts with lipid membranes in a protein receptor independent manner, as shown by the insertion of HAMLET into the membranes of giant unilamellar vesicles, disruption of the spherical shape and rapid tubulation, leading to a reduction in lumen size. (2) HAMLET creates a new membrane compartment for interactions with cellular targets and inhibits critical Ras oncogene driven signaling pathways. (3) This response to HAMLET does not occur in primary non-transformed cells, indicating tumor selectivity. The results suggest that HAMLET produces membrane conformations that serve as surrogate receptors for subsequent signal transduction, leading to tumor cell death. These effects are specific to the HAMLET complex, as the individual components of the complex, native alpha lactalbumin and oleic acid, did not affect the integrity of model membranes. The results indicate that both the partially unfolded protein and oleic acid are required to alter the structure of tumor cell membranes.

Ion fluxes regulate cellular homeostasis, through a multitude of effector functions. In parallel with the membrane changes, HAMLET activates rapid ion fluxes, with an influx of Na+ and Ca2+ and an efflux of K+ ions, as shown by real time confocal imaging, patch-clamping and fluorometry. The Na+ and K+ fluxes are thought to drive the cell death response, as ion flux inhibitors prevent HAMLET internalization, transcriptional responses and tumor cell death. These include amiloride, which inhibits the Na+/H+exchange and barium chloride (BaCl2), which inhibits K+ fluxes. HAMLET has not been shown to activate preformed ion channels, suggesting that channel-independent membrane permeabilization mechanisms might be involved.

Further mechanistic insights into this process have been gained, using synthetic alpha-helical peptides. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains were biologically active.

The results identify HAMLET as a membrane-perturbing agonist that triggers lethal ion fluxes in tumor cells.

2.3. Effects on nucleotide-binding proteins - kinases and GTPases

The ion fluxes activate a rapid p38 MAPK response as shown by transcriptomic analysis of HAMLET-treated tumor cells. In parallel, ERK1/2 phosphorylation is inhibited, consistent with the shift from proliferation to cell death. The activation of p38 and inhibition of ERK1/2 phosphorylation was reversed by ion flux inhibitors (amiloride or BaCl2), confirming the ion flux dependence. Importantly, pharmacological inhibitors of p38α and p38β delayed tumor cell death, as did p38-specific siRNAs. In contrast to tumor cells, normal differentiated cells showed weaker ion flux responses and less prominent changes in global transcription.

These findings suggested that affinity for conserved molecular motifs explains the apparent multitude of HAMLET targets in tumor cells. To identify such targets, we performed a proteomic screen of 8000 human proteins. By direct binding, we identified a large number of nucleotide binding proteins as HAMLET targets. These included 3 ATPases, 24 members of the Ras family of GTPases and 111 Kinases representing all branches of the kinome tree. In a kinase activity array, HAMLET acted as a pan-kinase inhibitor, reducing the activity of about 69% of the 476 kinases tested. This broad kinase inhibition was confirmed in protein lysates from HAMLET treated lung carcinoma cells, using an antibody microarray detecting phosphorylated proteins. Furthermore HAMLET was shown to co-localize with the Ras family of GTPases in membrane clusters and Ras activity was inhibited. The results identify HAMLET as an inhibitor of kinases and GTPases, to which tumor cells are addicted.

The results identify HAMLET as a potent and broad kinase inhibitor with specificity for tumor cells.

2.4. Chromatin interactions

In early studies our group has shown that HAMLET crosses the cytoplasmic membrane and accumulates in tumor cell nuclei. The accumulation of HAMLET in tumor cell nuclei was initially demonstrated by confocal microscopy of biotinylated HAMLET and fluorophore-labeled streptavidin and was confirmed using radiolabeled HAMLET. By far western blot and mass spectrometry analysis (MALDI-TOF), histones H2B, H3 or H4, were identified as nuclear HAMLET targets and HAMLET was shown to interfere with the formation of nucleosomes in the salt-jump assay. Furthermore, the sensitivity of tumor cells to HAMET is affected by chromatin acetylation, as histone deacetylase inhibitors open up the chromatin to HAMLET and synergistically kill tumor cells.

The results suggest that HAMLET may ‘seal the fate’ of dying tumor cells, through high affinity histone interactions and perturbation of the chromatin structure.

2.5. Proteasome inhibition

Proteasomes are crucial for extra-lysosomal protein degradation of endogenous misfolded proteins taking place in the barrel shaped 20S proteasome core. In a proteomic screen, catalytic proteasome subunits were identified as HAMLET targets. Furthermore, as α-lactalbumin is partially unfolded in the HAMLET complex, we hypothesized that the complex is targeted to the 20S proteasome for degradation. We found that HAMLET co-localizes with proteasomes in tumor cell cytoplasm and nuclei and detected an inhibitory effect of proteasome activity in whole cell extracts from HAMLET treated tumor cells. Interestingly, we also obtained structural evidence for proteasome disintegration by HAMLET, after incubation of HAMLET with intact 20S proteasomes, in vitro. Based on the above observations, we concluded that inhibition of proteasome activity might contribute to HAMLET-induced tumor cell death.

3. Structure of HAMLET complex

1 s2 0 s0006291x1631779x gr2

HAMLET is the first member in a new class of tumoricidal protein-lipid complexes, formed by partially unfolded α-lactalbumin and oleic acid (Fig. 2). α-Lactalbumin is the most abundant protein in human breast milk and the tightly packed globular conformation is stabilized by four disulfide bridges and a divalent calcium ion, with C- and N-terminal α-helical domains separated by a β-shee

t domain.

The native, globular state is defined by high affinity interactions with a strongly bound calcium ion (Ca2+), and c

onditions that release Ca2+, such as low pH or EDTA treatment are accompanied by a loss of tertiary structure definition. As a result the protein adopts a stable intermediate fold and forms a molten globule with loss of tertiary structure and retained secondary structure as demonstrated by near- and far-UV CD. In addition, ANS fluorescence is increased, reflecting the exposure of hydrophobic domains. Differences in surface topology have also been detected by limited proteolysis, compared to the native protein. In HAMLET, α-lactalbumin retains its partially unfolded characteristics even at physiological solvent conditions, suggesting that the binding of oleic acid stabilizes the protein in the partially unfolded state.

Moreover, HAMLET tolerates a certain degree of sequence variation, as purified α-lactalbumins from different species formed tumoricidal complexes with the fatty acid cofactor, oleic acid. This includes bovine, equine, caprine and porcine α-lactalbumins. The conversion yield for α-lactalbumin derived from other species is lower than for the human protein, however. 

1 s2 0 s0006291x1631779x gr3

Even though these proteins can form HAMLET-like complexes in vitro after purification and addition of the lipid, 

HAMLET-like complexes are not formed by low pH treatment of milk from other species. HAMLET formation has so far only been detect

ed in human milk, reflecting both the α-lactalbumin structure and the fatty acid composition.

The low-resolution structure of HAMLET has recently been solved using small angle X-ray scattering (SAXS). Th

e SAXS structure shows a two-domain conformation with a large globular domain and an extended C-terminal domain (Fig. 3). According to the SAXS structure, HAMLET exists as a monomer in solution with a molecular mass of 15 ± 2 kDa.

Unfolding of α-lactalbumin is not sufficient to achieve cytotoxicity. Mutant proteins that fail to fold to the native state are not cytotoxic for tumor cells. Tested protein variants include the high-affinity Ca2+ binding site mutant (D87A) and the fully r

educed cysteine-free mutant (rHLA All-Ala), in which all cysteine residues were substituted for alanines. The mutant proteins became cytotoxic after addition of the lipid cofactor, however. The lipid alone is significantly less cytotoxic than the HAMLET complex, at concentrations comparable to those present in the HAMLET complex.

3.1 Gain of function by loss of tertiary structure definition

This gain of tumoricidal activity by loss of 3D structural definition may appear paradoxical. The ‘one gene – one protein – one function’ paradigm considers the native state of a protein as the functional conformation, which is usually equated with the lowest free energy state of a given molecule. HAMLET is the first example of a protein that exhibits a well-defined function in its native state and then acquires a new and beneficial function after partial unfolding. Our findings suggest that a change in fold, in response to changing tissue environments, may allow a single polypeptide chain to exert vastly different, beneficial biological functions in different tissue compartments.

Complexes similar to HAMLET, which consist of protein and fatty acid constituents, exhibit cytotoxic activities. Examples of such complexes include BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumor cells), ELOA (Equine Lysozyme with Oleic Acid), and other oleic acid complexes with camel α-lactalbumin, β-lactoglobulin or pike parvalbumin. These findings suggest that the ability to form lipoprotein complexes may be a general feature of partially unfolded proteins.

4. Therapeutic and prophylactic effects of HAMLET

HAMLET has shown therapeutic efficacy in three cancer models; colon cancer, bladder cancer and a human glioblastoma xenograft model. In addition, HAMLET has been tested in a placebo-controlled study of human skin papillomas and in patients with bladder cancer. In each of these cases, we observed positive and interesting effects.

4.1. Human studies

a) Skin papillomas. We demonstrated therapeutic efficacy of HAMLET against human skin papillomas, in a placebo-controlled, blinded clinical study [44]. Patients with severe, therapy resistant papillomas on hands and feet received HAMLET or saline solution daily for 3 weeks. Topical application of HAMLET reduced the lesion volume by more than 75%. Moreover, a significant decrease in lesion volume was observed in all HAMLET treated patients and complete resolution of all lesions had occurred in about 83% of the HAMLET-treated patients after two years.

b) Bladder cancer. To examine if bladder cancer cells respond to HAMLET, patients with superficial bladder cancer received injections of HAMLET locally, into the bladder, on five consecutive days preceding bladder surgery. A rapid response was detected, with excretion of large numbers of tumor cells into the urine after two hours. Most of the cells were dead and showed evidence of apoptosis. By cystoscopy, a reduction in tumor size was detected at the time of surgery and apoptotic cells were seen in biopsy specimens.

4.2.  Animal models

a) Bladder cancer. Local instillation of HAMLET caused a reduction in tumor development, in mice with bladder cancer. Whole body fluorescence imaging showed that HAMLET is retained in tumor bearing mice compared to tumor free mice. Furthermore, five intra-vesical HAMLET instillations decreased tumor size and significantly delayed tumor development in tumor bearing mice compared to controls that received α-lactalbumin or phosphate buffer.

b) Glioblastomas. Therapeutic efficacy of HAMLET against human glioblastomas was investigated in ratsnu/nu after xenotransplantation of human glioblastoma cells. Local infusions of HAMLET delayed tumor development and prolonged survival. HAMLET penetrated throughout the tumor and triggered apoptosis in tumor cells. Importantly, there was no evidence of HAMLET toxicity for the normal brain.

c) Intestinal cancer. The therapeutic efficacy of HAMLET was investigated in a model of human colon cancer. APCMin/+ mice develop intestinal tumors that serve as a model of human disease. APC mutations occur in the majority of patients with colon cancer and in families with inherited susceptibility to colon cancer. Peroral administration of HAMLET caused a significant reduction in tumor size and polyp number in this model. In addition, HAMLET accumulated specifically in tumor tissue. In parallel, the expression of key oncoproteins was reduced after HAMLET administration, including β-catenin, Ki67, COX2 and VEGF. By whole genome transcriptomic analysis HAMLET was shown to inhibit the expression of genes in the Wnt signaling pathway in APCMin/+ mice. Furthermore, we supplied HAMLET into the drinking water of young mice for ten weeks, from the time of weaning. This treatment reduced tumor development by 60%, suggesting that HAMLET acts prophylactically.

In conclusion, these studies have established that local HAMLET administration is effective, with therapeutic and prophylactic effects against several different tumors. Importantly, we did not observe toxic effects on healthy tissues in treated patients or animals.

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 drivenas 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.

For more Information please visit HamletPharma.com

Publication list:

Authors

Title

Journal

Ho, J., Nadeem, A., Svanborg, C.

HAMLET – A protein-lipid complex with broad tumoricidal activity

Elsevier, Biochemical and Biophysical Research Communications, Volume 482, Issue 3, 15 January 2017, Pages 454–458

Chaudhuri A, Prasanna X, Agiru P, Chakraborty H, Rydström A, Ho JC, Svanborg C, Sengupta D, Chattopadhyay A. 2016. 

Nature Scientific Reports. 6:35015. doi: 10.1038/srep35015.

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 1

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