Peptides for blocking IL1RAP protein-protein interaction and uses thereof for treatment of disease

Abstract

Methods and agents are provided for inhibiting interleukin 1 receptor accessory protein (IL1RAP) protein-protein interaction to treat a broad spectrum of diseases and conditions.

Claims

1. A method of treating a disease or condition in a subject, to inhibit interleukin 1 receptor accessory protein (IL1RAP) interaction with interleukin 1 receptor 1 (IL1R1) or interleukin 1 receptor 2 (IL1R2), the method comprising administering to the subject an agent comprising a peptide in an amount effective to inhibit IL1RAP interaction with IL1R1 or IL1R2, wherein the peptide has 18-30 amino acids and comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7, wherein the disease or condition is chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), asthma, allergy, anaphylaxis, cardiovascular disease, or arthritis.

2. The method of claim 1, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

3. The method of claim 1, wherein the peptide is a synthetic peptide.

4. The method of claim 1, wherein the peptide is conjugated to a cytotoxic agent, a polyethylene glycol (PEG) or a nanoparticle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1B. In silico design of IL1RAP peptides of interference. A) Crystal structure of the extracellular domains of the IL1R1 (sphere representation) bound to IL1RAP (ribbon representation) (PDB ID: 4DEP) is depicted with the aminoacids at the receptor-coreceptor contact interphase (sphere representation) B) structural superimposition of 4DEP and 3O4O.

(2) FIG. 2. Sequence alignment of IL1R1 and IL1R2. Amino acid sequence alignment of human IL1R1 (SEQ ID NO:4) with human IL1R2 (SEQ ID NO:5) using ClustalW.sup.27, 28. Strictly conserved residues, conserved substitutions (according to the ClustalW defined strong groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY or FYW) and partially conserved substitutions are highlighted (according to the clustalw defined weak groups: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM or HFY). The filled circles above and below the aligned sequences indicate, respectively, the residues involved in IL1R1-IL1RAP and IL1R2-IL1RAP interactions within 4.5 angstroms.

(3) FIG. 3A-3C. IL1RAP peptides of interference reduce cell growth. A) Image of the crystal structure of the extracellular domains of the IL1RAP (sphere representation) bound to IL1R1 (ribbon representation) (PDB ID:4DEP) is depicted with peptide of interference P1 (sphere representation). B) Scheme of the IL1RAP peptides of interference with the respective amino acid positions indicated with numbers. Peptide 1 (p1) (SEQ ID NO:1), Peptide 2 (p2) (SEQ ID NO:2) C) THP-1 cells were exposed to scrambled peptide (scrambled), or IL1RAP peptides (P1 and P2) at the indicated concentrations or left untreated (dashed line) and evaluated for cell viability determined by trypan blue staining and manual counts at 12 and 24 hours. Error bars represent the standard error of the mean of two independent experiments.

(4) FIG. 4. IL1RAP peptides of interference reduce cell growth in two different leukemia cell lines. THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) (upper panel) and HL60 cells (human promyelocytic leukemia cells) (lower panel) were exposed to scrambled peptide (scrambled), or IL1RAP peptides (P1 and P2) at 300 μM concentrations or left untreated and evaluated for cell viability determined by trypan blue staining and manual counts at the indicated durations of exposure. Error bars represent the standard error of the mean of two independent experiments.

(5) FIG. 5A-5B. A) IL1RAP blockade causes reduced clonogenic potential of AML cells. THP-1 cells were exposed to 300 μM IL1RAP peptides (P1 and P2), scrambled control peptide (scrambled) or left untreated (UT) and evaluated for colony formation capacity in methylcellulose. The results are expressed as total number of colonies/1000 plated cells. Error bars represent the standard deviation of two replicates. Furthermore, primary human AML cells from patients were treated with peptides and evaluated in the same way (FIG. 5B). B) IL1RAP blockade causes reduced clonogenic potential of primary AML mononuclear cells. AML mononuclear cells derived from peripheral blood were exposed to 300 μM IL1RAP peptides (P1 and P2), scrambled control peptide (scrambled) or left untreated (UT) for 4 hours and evaluated for colony formation capacity in methylcellulose. The results are expressed as total number of colonies/50,000 plated cells. Error bars represent the standard deviation of two replicates.

(6) FIG. 6. IL1RAP peptides of interference do not reduce clonogenic potential of healthy bone marrow mononuclear cells (BMMNC). BMMNC were exposed to scrambled peptide (scrambled), or IL1RAP peptides (P1 and P2) for 4 hours at 300 μM concentrations or left untreated (UT) and evaluated for colony formation capacity in methylcellulose. Colonies were scored at 14 days. The results are expressed as total number of colonies/5×10.sup.4 plated cells. Error bars represent the standard deviation of two replicates.

(7) FIG. 7. IL1RAP blockade causes apoptosis and cell cycle arrest in G2 of IL1RAP dependent human leukemia cells. THP1 cells were exposed to 300 μM IL1RAP peptides (P1 and P2), scrambled control peptide (scrambled), peptide buffer (vehicle) or left untreated (UT) and evaluated for cell cycle progression determined by flow cytometry.

(8) FIG. 8. IL1RAP blockade causes cell death of IL1RAP dependent human leukemia cells. THP1 cells were exposed to 300 μM IL1RAP peptides (P1 and P2), scrambled control peptide (scrambled), peptide buffer (vehicle) or left untreated (UT) and evaluated for cell viability determined by Annexin V/DAPI staining and flow cytometry. The results are expressed as percentage of total cells.

(9) FIG. 9. IL1RAP blockade changes in cell morphology of IL1RAP dependent human leukemia cells. THP1 cells were exposed to 100 μM IL1RAP peptide P2, scrambled control peptide (scrambled), peptide buffer (vehicle) or left untreated and evaluated for cell morphology.

(10) FIG. 10. IL1RAP peptides of interference. THP1 cells were exposed to increasing concentrations of IL1RAP peptides P1 (upper panel) and P2 (lower panel), scrambled control peptide (scrambled) or left untreated (UT) and evaluated for changes in phospho-IRAK1, phospho-FLT3 and phospho-STAT3 by western blot.

(11) FIG. 11. Bar graphs show an Image J quantification of western blots from FIG. 10. Dotted line indicates the level of untreated sample. Values are expressed as fold-change to untreated sample.

DETAILED DESCRIPTION OF THE INVENTION

(12) The invention provides a method of treating a disease or condition in a subject in which it is desirable to inhibit interleukin 1 receptor accessory protein (IL1RAP) protein-protein interaction, the method comprising administering to the subject an agent comprising a peptide in an amount effective to inhibit IL1RAP protein-protein interaction.

(13) The invention also provides an agent for treating a disease or condition in a subject in which it is desirable to inhibit interleukin 1 receptor accessory protein (IL1RAP) protein-protein interaction, the agent comprising a peptide that inhibits IL1RAP protein-protein interaction.

(14) The disease or condition can be, for example, one or more of cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), papillary thyroid carcinoma, infection, asthma, allergy, anaphylaxis, cardiovascular disease, arthritis, neurodegenerative disease, and inflammatory disease.

(15) The peptide can target, for example, conserved residues of a common interface of IL1RAP that interacts with interleukin 1 receptor 1 (IL1R1), interleukin 1 receptor 2 (IL1R2), interleukin 1 receptor-like 1 (IL1RL1), interleukin 1 receptor-like 2 (IL1RL2), signal-regulatory protein alpha (SIRPA), ST2, KIT, Fms-like tyrosine kinase 3 (FLT3) (including its mutant variants, e.g. FLT3-internal tandem duplication, or FLT3 juxtamembrane mutants), macrophage colony-stimulating factor receptor (MCSFR), toll-like receptor (TLR), or other proteins interacting through this molecular interface.

(16) The peptide can, for example, comprise 18-30 amino acids that include one of the following amino acid sequences: AGDKDRLIVMENKPTRPV (SEQ ID NO:1), AGDKDRLIVMENKPTHGID (SEQ ID NO:2), AGDKDRLIVMENKPTXXX (SEQ ID NO:6) or AGDKDRLIVMENKPTXXXX (SEQ ID NO:7). The peptide can, for example, consists of amino acid sequence AGDKDRLIVMENKPTRPV (SEQ ID NO:1) or AGDKDRLIVMENKPTHGID (SEQ ID NO:2).

(17) The invention further provides a synthetic peptide consisting of amino acid sequence AGDKDRLIVMENKPTRPV (SEQ ID NO:1) or amino acid sequence AGDKDRLIVMENKPTHGID (SEQ ID NO:2).

(18) The peptides can be conjugated to different agents, for example, a cytotoxic agent, a polyethylene glycol (PEG) or a nanoparticle. The cytotoxic agent can be, for example, a small molecule of 2,000 daltons or less, or 1,000 daltons or less, or 500 daltons or less. The PEG can have a molecular weight, for example, of 200-2,000 daltons or more. Preferably the nanoparticle is between 1 and 100 nanometers in size.

(19) The invention still further provides a method of screening for a candidate agent for treating cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), papillary thyroid carcinoma, infection, asthma, allergy, anaphylaxis, cardiovascular disease, arthritis, neurodegenerative disease, and/or inflammatory disease, the method comprising testing whether the agent inhibits interleukin 1 receptor accessory protein (IL1RAP) protein-protein interaction, wherein an agent that inhibits IL1RAP protein-protein interaction is a candidate agent for treating cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), papillary thyroid carcinoma, infection, asthma, allergy, anaphylaxis, cardiovascular disease, arthritis, neurodegenerative disease, and/or inflammatory disease.

(20) The agents or peptides can be administered to subjects using routes of administration known in the art. The administration can be systemic or localized to a specific site. Routes of administration include, but are not limited to, intravenous, intramuscular, intrathecal or subcutaneous injection, oral or rectal administration, and injection into a specific site.

(21) The invention further provides a pharmaceutical composition comprising any of the agents or peptides identified herein and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” is (i) compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and (ii) suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions.

(22) This invention will be better understood from the Experimental Details that follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

(23) Introduction and Overview

(24) Specific peptides were designed to interfere with the oligomerization of interleukin 1 receptor accessory protein (IL1RAP) with its receptors partners (including KIT, and FLT3, known key drivers of AML pathogenesis, which the inventors recently identified to interact with IL1RAP in AML cells (unpublished). These peptides enable specific interruption of IL1RAP mediated signaling representing a fundamentally novel therapeutic approach. As an example, interfering peptides were shown to lead to inhibition of downstream signaling and significant growth inhibition of IL1RAP-dependent leukemia cells.

(25) Materials and Methods

(26) Peptide Design.

(27) To interrupt the interaction between the extracellular domains of IL1RAP and its associated molecules small peptides were designed utilizing the crystal structures of the extracellular complexes of IL1RAP, IL1R1 and IL1β (PDB ID: 4DEP.sup.19) and IL1RAP, IL1R2 and IL1β (PDB ID: 3O4O.sup.20) available in the Protein Data Bank (PDB).sup.21. Contacts between amino acids from two different polypeptide chains (IL1RAP against IL1R1 and IL1R2) were defined as having any two atoms at a maximum distance of 4.5 Angstroms, which sufficiently encompasses Van der Waals, hydrogen bond, and most hydrophobic interactions. Structural superimposition between the two complexes was carried out using the program STAMP (STructural Alignment of Multiple Proteins).sup.22 as made available in the program VMD (Visual Molecular Dynamics).sup.23.

(28) Cell Lines.

(29) The AML cell line THP-1 was grown in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 0.05 mM betamercaptoethanol and 1% penicillin/streptomycin.

(30) Peptide Resuspension and Cell Line Treatment.

(31) Two specific peptides (P1: AGDKDRLIVMENKPTRPV (SEQ ID NO:1) and P2: AGDKDRLIVMENKPTHGID (SEQ ID NO:2)) and one scrambled control peptide (LKDMPGRPNERDKVAlTV (SEQ ID NO:3)) were synthesized by Genscript (Piscataway, N.J.) using stepwise SPPS chemical methods and purified by HLPC (purity >98%). Peptides were free of trifluoroacetate. Lyophilized peptides were aliquoted and resuspended in 0.15M PBS pH 7.0 before use. THP-1 cells were seeded at 0.5×10.sup.6 cells/ml and treated with control, vehicle or specific peptides at the indicated concentrations every 4 hours during the indicated periods of time.

(32) Cell Viability and Clonogenic Assays.

(33) Cell viability was assessed at the indicated time points by trypan blue staining and manual cell counts. Viable cells were plated in methylcellulose (StemCell technologies H4434, or R&D HSC002SF) at 1000 cells/ml in 6-well plates for cell lines and at 5×10.sup.4 cells/ml for bone marrow mononuclear cells (BMMNC). Cells were incubated at 37° C. and 5% CO.sub.2. Colonies were scored after 7 days or 14 days in culture for cell lines or primary cells, respectively.

(34) AML Clonogenic Assays.

(35) Primary AML mononuclear cells derived from peripheral blood were seeded in cellgro media with cytokines (FLT3L 300 ng/ml, TPO 100 ng/ml, SCF 300 ng/ml and IL3 60 ng/ml) and antibiotic. Cells were treated with IL1RAP peptides of interference or scrambled control at 300 uM for 4 hours. After that period, cell viability was assessed by trypan blue staining and manual cell counts. Viable cells were plated in methylcellulose (StemCell technologies H4434, or R&D HSC002SF) at 5×10.sup.4 cells/ml in 6-well plates. Cells were incubated at 37° C. and 5% CO.sub.2. Colonies were scored after 14 days in culture.

(36) Flow Cytometric Determination of Cell Death.

(37) In order to determine viability after peptide treatment, 1×10.sup.4 THP-1 cells were washed with PBS and mixed with pre-diluted PE-conjugated Annexin V (BD Pharmigen) and DAPI. Cells were stained at room temperature for 15 minutes and suspended in 0.2 ml of Annexin V-FLUOS incubation buffer (Roche) for analysis.

(38) Cell Cycle Analysis.

(39) Cell cycle analysis was performed by staining with propidium iodide (PI). In brief, 1×10.sup.6 THP-1 cells after treatment with peptides were rinsed with PBS, fixed Fixation Buffer (BD Cytofix™ fixation buffer) for 30 min at 4° C. 5 ml of cold 70 to 80% ethanol were added to the cells while vortexing and the suspension was stored overnight at −20° C. Cells were washed twice (first with PBS and then with 1×PBS, 2% FBS) and spun down for 10 minutes at 1,500 rpm. Cell pellet was resuspended in 0.5 mL of PI/RNase Staining Buffer (BD Pharmingen® PI/RNase staining buffer) and incubated 15 minutes at room temperature and immediately analyzed by flow cytometry using a FACSAria II Special Order System (BD Biosciences, San Jose, Calif.).

(40) Cell Morphology.

(41) 1×10.sup.4 THP-1 cells after treatment with peptides were rinsed with PBS and 0.1 ml of cell suspension was cytospun onto polylysine coated slides at minimum speed for 4 minutes in a cytocentrifuge (StatSpin Cytofuge). Slides were stained according to manufacturer's protocol using the Romanowsky staining Diff-Quick (IMEB) kit. Cells were imaged with an Axiovert 200M microscope (Zeiss) and Olympus SZ61 microscope.

(42) Preparation of Cell Lysates and Immunoblotting.

(43) 3×10.sup.5 THP-1 cells after treatment with peptides, were lysed with modified RIPA buffer (Tris-HCl 50 mM pH7.4, NP-40 1%, Na-deoxycholate 0.25%, NaCl 150 mM, EDTA 1 mM, PMSF 1 mM, Roche protease inhibitor cocktail Mini complete 1×, Na.sub.3VO.sub.4 1 mM, NaF 1 mM and β-glycerophosphate 20 mM). The lysate was incubated in orbital shaker 4° C. 15-30 min. in a cold room. Then, cells were spun at 14,000×g 4° C. for 15 min and the supernatant was transferred to a pre-cooled tube. Protein concentration in cell lysate was determined, 25 μg of lysate were mixed with 2×SDS loading buffer, boiled for 5 min, and loaded in 8% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane and analyzed by western blot with indicated antibodies. Membranes were stripped as needed using a previously published protocol.sup.24. For immunoblotting, the following antibodies were used: phospho-FLT3 (Tyr591) Mouse monoclonal antibody (Cell signaling #3466), anti-phospho IRAK (T209-abcam 61799), phospho-STAT3 Tyr705) antibody (Cell signaling #9131) and Actin Antibody (C-11 sc-1615). Membranes were developed using Pierce ECL Western Blotting Substrate (thermo) or SuperSignal West Femto Chemiluminescent Substrate (thermo).

(44) Results and Discussion

(45) Design of IL1RAP Inhibitory Peptides.

(46) The development of small molecules that can specifically disrupt IL1RAP-receptor interactions will be a valuable tool not only to target IL1RAP-mediated signaling in AML, MDS, and other diseases, but it will also facilitate study of IL1RAP protein-protein interactions. Therefore, studies were designed to interrupt the interaction between the interacting domains of IL1RAP and its associated molecules with small peptides of interference.

(47) Crystallographic data were used to design a peptide to occupy the interaction surface in the extracellular domain of the IL1RAP-IL1R1 as well as the IL1RAP-IL1R2 complexes (FIG. 1A). Available in the Protein Data Bank (PDB).sup.21 are the structures of the interacting extracellular domains of IL1RAP in complex with IL1R1 (PDB ID: 4DEP.sup.19) and with IL1R2 (PDB ID: 3O4O.sup.20). The complex in 4DEP contains 3 polypeptide chains: 1) IL1β as chain A, covering residues 1-151, 2) IL1RAP as chain B, covering residues 7-310 and 3) IL1R1 as chain C, covering residues 3-326. Similarly, the complex in 3O4O contains 3 polypeptide chains: 1) IL1β as chain A, covering residues 1-152, 2) IL1R2 as chain B, covering residues 4-326 and 3) IL1RAP as chain C, covering residues 5-310. The crystal structure of the complex in 4DEP or 3O4O reveals that the largest interface is formed between IL1R1 and IL1β (similarly for IL1R2), with an average of 41 residues involved. Such a large interface is produced by the wrapping of IL1β by the two flexibly linked immunoglobulin domains of IL1R1. IL1RAP also physically interacts with the IL1β polypeptide, averaging 15 residues at the contact area. The contact interface between IL1RAP and either IL1R1 or IL1R2 is formed by 16 residues on average.

(48) The above mentioned complexes are superimposable (see methods) (FIG. 1B). The structural overlay reveals that IL1RAP utilizes the same interface to interact with both receptors IL1R1 and IL1R2. Closer scrutiny of the receptor-coreceptor interface exposes the particular residues of IL1RAP and IL1R1/IL1R2 that are in contact (FIG. 1A). These contacting residues between IL1RAP and its receptors are also highlighted on the sequence alignment in FIG. 2. Most of the residues from IL1R1 and IL1R2 in contact with IL1RAP are conserved or at least semi-conserved (with a few exceptions). Out of 12 aligned residues within 4.5 angstroms, 4 are identical, 5 conserved (meaning that general properties of the residue side-chains are conserved), 1 semi-conserved (D120-5134 who only share the characteristic of being polar) and 2 unrelated substitutions.

(49) The design of peptides of interference was based on the idea that they should resemble the residue identities in the interaction surface of IL1RAP and its receptors. Therefore, residues found at the interaction surface of IL1R1 or IL1R2 with IL1RAP were considered in the design of the peptides. A linear path was found in the structures that traverses most of the common interface residues; peptides were designed that combine most of these common contacts (FIG. 3A). This strategy is potentially powerful because the designed peptides would simultaneously interrupt both interactions, and including interactions with other molecules.

(50) IL1RAP Peptides of Interference Suppress Growth of AML Cells.

(51) Since knockdown of IL1RAP with lentivirally expressed shRNAs in THP-1 cells reduced cell survival.sup.6, it was determined whether specific or scrambled control peptides affected cell viability in the IL1RAP-positive cell line THP-1. THP-1 cells were treated with either different peptides of interference (P1 and P2 in FIG. 3B) or scrambled control peptide, or the cells were left untreated. Peptides were added at 4 hour intervals, for a period of 12 hours. Specific peptides P1 and P2, but not control peptide, caused loss of viability and growth suppression of THP1 cells (FIG. 3C, 4). In addition, IL1RAP peptides of interference P1 and P2 led to inhibition of leukemic colony formation of THP-1 cells in comparison to the scrambled control peptide (FIG. 5).

(52) IL1RAP Peptides of Interference do not Suppress Growth of Healthy Bone Marrow Mononuclear Cells.

(53) In contrast to the effects of peptides P1 and P2 on AML cells, no inhibitory effects of these peptides were observed on healthy control bone marrow mononuclear cells (FIG. 6). These include healthy stem and progenitor cells that do not express IL1RAP..sup.6

(54) IL1RAP Peptides of Interference Induced Apoptosis and/or Cell Cycle Arrest in AML Cells.

(55) The phenotypic effects of the IL1RAP interaction blockade in AML cells were assessed in vitro in THP-1 cells. THP-1 cells treated with 300 μM of either IL1RAP peptides P1 or P2 or control peptide were stained with Annexin V and DAPI and subjected to flow cytometric analysis. Changes in cell cycle were also analyzed by Propidium iodide staining and flow cytometry. THP-1 cells underwent a moderate cell cycle arrest in G2 (FIG. 7) in the presence of IL1RAP peptide P2 but not control peptide (scrambled) or peptide buffer (vehicle). At 24 hours after initial treatment, IL1RAP specific peptides P1 and P2 caused an increase of apoptosis as determined by Annexin V/DAPI staining (FIG. 8). THP-1 cells treated with IL1RAP blocking peptides demonstrated a higher degree of cell death (DAPI.sup.+/Annexin V.sup.+); 39% (P2) and 35% (P1) of cells were considered apoptotic upon IL1RAP blockade. IL1RAP peptides P1 and P2 showed an overall reduction in viability (DAPI.sup.+/Annexin V.sup.+) of 28% (P2) and 14% (P1) in comparison to the scrambled control peptide. In addition PI staining showed an increase of subG0 for IL1RAP peptide P2 that indicates increased levels of apoptosis (FIG. 7).

(56) IL1RAP Peptides of Interference Induced Changes in Morphology Indicative of Differentiation of AML Cells.

(57) THP-1 monocyte cells can differentiate into macrophage-like cells. During the process of differentiation cells become adherent and adjust their morphology. The THP-1 cells that remained viable after 24 hours of treatment with P1 and P2 showed substantial changes in morphology which could be indicative of monocytic differentiation (FIG. 9). In contrast, cells treated with scrambled peptide maintained a blast-like morphology (FIG. 9).

(58) IL1RAP Peptides of Interference Affect IL1 and FLT3 Signaling in AML Cells.

(59) In active IL1 signaling, the interaction of IL1RAP with IL1R1 leads to the recruitment and phosphorylation of the interleukin 1 receptor associated kinase 1 (IRAK1). To assess whether IL1RAP peptides of interference were effective in blocking IL1 signaling, the phosphorylation levels of IRAK1 were determined 48 hours after treatment with IL1RAP specific peptides or scrambled control. A profound concentration dependent reduction of phospho-IRAK1 was observed in THP-1 cells treated with IL1RAP peptide P1 in comparison with scrambled control peptide (FIGS. 10, 11). In addition, both IL1RAP specific peptides P1 and P2 showed reduced phosphorylation levels in phospho-FLT3, a receptor tyrosine kinase that also interacts with IL1RAP in leukemia cells (Barreyro unpublished results). It is possible that IL1RAP peptides of interference P1 and P2 have different binding affinity because similar changes were not observed in phosphorylation of IRAK1 at 48 hs. In addition, levels of phospho-STAT3, a transcription factor that is activated by IL1 and FLT3 signaling.sup.25,26, changed at 48 hs after the initial treatment with IL1RAP peptide P2 to a greater extent than with IL1RAP peptide P1 (FIGS. 10, 11). This finding suggests that IL1RAP peptides of interference successfully block IL1 and FLT3 signaling and attenuate downstream activation of signal transducer and activator of transcription STAT3 through the interleukin (IL)-1 and FLT3 signaling pathways by blocking IL1R1/IL1RAP and IL1RAP/FLT3 receptor interactions in leukemic cells.

(60) The present invention provides a functional interference strategy to inhibit IL1RAP function, independent of current antibody-based strategies which use IL1RAP solely as a surface label to trigger/activate an immune response against IL1RAP-expressing cells, independent of whether IL1RAP plays an actual functional role for these cells.

(61) In contrast to the present invention of using peptides of interference to block IL1RAP protein-protein interaction, antibody-based approaches are thought to mediate anti-tumor efficacy via antibody-dependent cellular cytotoxicity (ADCC) via NK cell-mediated induction of cell death. Unlike the present approach, antibody-based approaches are likely to have limited therapeutic efficacy in patients with diseases such as AML and MDS who are immunocompromised and will have impaired ADCC.

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