METHODS FOR PREDICTING THE RESPONSIVENESS OF A PATIENT AFFECTED WITH MALIGNANT HEMATOLOGICAL DISEASE TO CHEMOTHERAPY TREATMENT AND METHODS OF TREATMENT OF SUCH DISEASE

Abstract

The present invention relates to antagonists of GDF11, for use in the treatment of malignant hematological disease, such as Acute Myeloid Leukemia (AML). The present invention also relates to a method for predicting the responsiveness of a patient affected with malignant haematological disease, such as Acute Myeloid Leukemia, to a chemotherapy treatment.

Claims

1. A method for predicting the responsiveness of a patient affected with a malignant haematological disease to a chemotherapy treatment, comprising measuring a level of GDF11 in a blood sample from said patient and comparing the level of GDF11 with a control reference value, wherein if the level of GDF11 in the patient is inferior to the control reference value it is concluded that the patient is a responder to the chemotherapy treatment.

2. The method according to claim 1, wherein the malignant haematological disease is acute myeloid leukemia.

3. The method according to claim 1, wherein the chemotherapy treatment is selected from the group consisting of daunorubicin, cytarabine, mitoxantrone, decitabine, and azacitidine.

4. (canceled)

5. A method for monitoring the effectiveness of treatment of a patient affected with a malignant haematological disease to an anti-cancer treatment comprising a step of measuring a level of GDF11 in a blood sample from said patient and comparing the level of GDF11 with a control reference value.

6. The method according to claim 5, wherein the anti-cancer treatment is chemotherapy treatment and/or a GDF11 antagonist.

7-13. (canceled)

14. A pharmaceutical composition comprising a GDF11 antagonist and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14 further comprising an additional therapeutic agent.

16. A method of preventing or treating a malignant haematological disease in a subject comprising administering to the subject a therapeutically effective amount of a GDF11 antagonist.

17. The method of claim 16, wherein the GDF11 antagonist is an inhibitor of the interaction between GDF11 and GDF11 receptor (ActRIIA or ActRIIB).

18. The method according to claim 16, wherein said antagonist is a soluble GDF11 receptor polypeptide.

19. The method according to claim 16, wherein said antagonist is an anti-GDF11 neutralizing antibody or aptamer.

20. The method according to claim 16, wherein said antagonist is an inhibitor of GDF11 gene expression,

21. The method of claim 20, wherein said inhibitor of GDF11 gene expression is a small inhibitory RNA (siRNA), a nuclease, a ribozyme, or an antisense oligonucleotide.

22. The method according to claim 16, wherein the malignant haematological disease is acute myeloid leukemia.

23. The method according to claim 16, wherein the subject suffers from a drug resistant tumor or tumor relapse.

24. A method for treating a patient affected with a malignant haematological disease, comprising i) identifying the patient as a chemotherapy responder by measuring a level of GDF11 in a blood sample from said patient, comparing the level of GDF11 with a control reference value, and determining that the level of GDF11 is inferior to the control reference value, and ii) treating the patient with chemotherapy.

Description

FIGURES

[0193] FIG. 1: gdf11 mRNA Levels are Higher in AML Compared with Normal HSC or Controls.

[0194] (A) Retrospective transcriptomic analysis of datasets GSE 13159 and GSE17054 show that gdf11 expression is higher in AML subgroups presenting recurrent cytogenetic alterations (AML with AML1-ETO n=39, AML with inv 16 n=28, APL n=37, AML with 11q23 n=38) than in normal hematopoietic stem cells (HSC, n=8), p<10-4. (B) gdf11 expression is higher in AML (n=404) than in non AML control patients (n=138)(GSE 15061), p<10-4.

[0195] FIG. 2: GDF11 is overexpressed in a sub-population of patients with AML.

[0196] (A) Serum GDF11 concentrations in AML patients (n=195) versus controls (n=55), means+/SEM. (B) Serum GDF11 concentrations>7 in AML patients (n=29) and controls (n=14), means+/SEM.

[0197] FIG. 3: GDF11 levels are not correlated with full blood count parameters in AML.

[0198] Hemoglobin levels (A), platelets (B) and absolute blast cells counts (C) were correlated with GDF11 in 140 AML patients enrolled in the LAMIR2006 clinical trial.

[0199] FIG. 4: GDF11 overexpression is not associated with a recurrent genotype.

[0200] Prevalence of the most frequent recurrent mutations in 132 AML patients with low (<40 pg/mL) or elevated (40 pg/mL) GDF11 levels.

[0201] FIG. 5: GDF11 overexpression is associated with induction therapy failure and primary chemoresistance.

[0202] (A) Patients with elevated GDF11 level (40 pg/mL, n=18) harbor at early response assessment (D15) after induction therapy more frequently blastic bone marrow compared to patients with lower GDF11 (n=148; P=0.016). (B) Patients with elevated GDF11 level (n=16) need more chemotherapy courses to obtain complete remission (P=0.001).

[0203] FIG. 6: GDF11 blockade induces apoptosis and growth arrest in AML cell lines.

[0204] (A) Dose-response apoptosis in 4 AML cell lines (MOLM 14, THP1, HL 60 and MV 4-11). (B) Uptiblue-proliferation assay in MV4-11 and THP1 cell lines treated with anti GDF11 antibody (ab71347). (C) Dose-response apoptosis in MV4-11 AML cell line with 4 anti-GDF11 antibodies.

[0205] FIG. 7: GDF11 blockade induces apoptosis and growth arrest in primary AML cell.

[0206] Primary AML cells from eight different patients (SU 1 to 8) were cultured with growing concentrations of anti GDF11 antibody (ab71347).

EXAMPLE

[0207] Material & Methods

[0208] Patients and Controls

[0209] Sera and blast cells from patients with AML were obtained from the GOELAMS' biobank after acceptance of the study by the scientific committee. 174 patients enrolled in the LAMIR2006 clinical trial (patients aged between 18-60 years and presenting de novo AML with intermediate risk, NCT00860639) and 21 patients enrolled in the CBF trial (patients aged between 18-60 years and presenting de novo or therapy-related Core Binding Factor (CBF) AML, NCT00428558) were included in this study. 55 sera were obtained from healthy controls after informed consent.

[0210] Statistics

[0211] All statistics were performed with GraphPad Prism 5 (GraphPad Software Inc.) All tests used were two-tailed tests and a P-value<0.05 was considered as significant.

[0212] Cell Culture and Cytotoxicity Assays

[0213] MOLM-14, MV4-11, HL60, THP1 AML cells lines and primary AML cells were cultured in alpha MEM medium supplemented with 5% FCS and glutamine. Cell lines were cultured at 250 000/ml in 24-well plates during 48h with various concentrations of anti-human GDF11 antibodies: ab71347, ab56645 (Abcam), WH0010220M3 (Sigma) and sc-6884 (Santa Cruz Biotechnology). Primary AML cells were cultured at 3 10.sup.6/ml in 48-well plates during 5 days. Apoptosis was measured with Annexin V-FITC apoptosis detection kit (Becton Dickinson Pharmingen) coupled with TO-PRO-3 (Life Technologies) staining according to manufacturers' instructions. For Uptiblue cytotoxicity bioassay (Interchim), cells were cultured at 50 000/ml in 96-well plates during 48h and fluorescence was measured with a Victor X4 multilabel plate reader (Perkin Elmer).

[0214] Elisa

[0215] GDF11 levels were measured using Elisa kit (Mybiosource) according to manufacturer's instructions.

[0216] Results

[0217] GDF11 is Overexpressed in a Sub-Population of Patients with AML.

[0218] We investigated the levels of GDF11 expression in AML patients. Through an investigation of distinct AML subgroups presenting recurrent cytogenetic alterations found in the disease (http://servers.binf.ku.dk/hemaexplorer) we found that gdf11 expression was higher in AML with AML1-ETO, APL, AML with inv(16)/t(16;16) and AML with t(11q23) subgroups compared to healthy hematopoietic stem cells (FIG. 1A; p<10-4). Further retrospective analysis of transcriptomic data from a large set of AML patients (GSE 15061) revealed that gdf11 expression was increased in AML patients compared to non-AML controls (FIG. 1B, p<10-4). Therefore, gdf11 levels are increased in samples from AML compared to healthy normal HSCs or non-AML controls.

[0219] To further confirm results from gene expression analysis we searched to detect circulating GDF11 levels in favorable and intermediate cytogenetic risk-AML patients at diagnosis (n=195 sera from the GOELAMS' biobank-Groupe Ouest-Est des Leucemies Aigues et Maladies du Sangprotocols NCT00860639 and NCT00428558). Healthy individuals with no declared hematological disorder were used as controls (n=55 sera). GDF11 levels were determined by a commercial ELISA kit (Mybiosource).

[0220] Detectable GDF11 levels (>7pg/ml) were found in 16% AML patients and did not differed in frequency from healthy subjects (FIG. 2A). Absence of differences between the frequencies of individuals with detectable GDF11 levels suggests that GDF11 expression is not a feature of AML. When comparing subject groups presenting detectable GDF11 (>7pg/ml) observed that the mean level of circulating GDF11 was significantly elevated in AML patients (7613; range, 8-294 pg/mL) compared with healthy controls (446; range, 8-95 pg/mL) (p=0.035; FIG. 2B). Therefore, circulating GDF11 levels are increased in AML patients compared to controls.

[0221] GDF11 Overexpression is not Associated to Anemia of AML or Disease Burden

[0222] AML patients frequently develop anemia which can be associated with alterations of erythroid lineage differentiation. Since increased GDF11 levels have been shown to promote ineffective erythropoiesis in both MDS and thalassemia.sup.18 We asked whether GDF11 overexpression could be associated with anemia of AML. Analysis of sera obtained from intermediate cytogenetic risk-AML patients at diagnosis (N=140) with different degrees of anemia or normal hemoglobin levels (hemoglobin levels ranging from 6 to 14 g/L) shown that there was no correlation between anemia and circulating GDF11 levels (FIG. 3A). Further analysis showed the absence of correlation between circulating GDF11 levels and thrombocytopenia (FIG. 3B) or the number of circulating blasts (FIG. 3C) suggesting that GDF11 is not correlated with disease progression leukemic burden.

[0223] GDF11 Overexpression does not Correlates with Common AML Somatic Mutations Found in NK-AML

[0224] Since increased GDF11 serum levels were present in a subpopulation of intermediate cytogenetic risk AML patients which are mainly normal karyotypes (80%) we searched for correlations between increased circulating levels of GDF11 and recurrent somatic mutations present in this patient group. Elevated circulating GDF11 levels were not associated with FLT3, NPM1, CEBPa, ASXL1, IDH1, IDH2, WT1 or DNMT3A mutations (FIG. 4). Therefore, GDF11 overexpression is independent of mutational status of AML patients.

[0225] GDF11 Overexpression Correlates with Impaired Remission Induction.

[0226] We next searched for the correlation of increased GDF11 levels and response to induction chemotherapy. Whereas patients presenting circulating GDF11 levels of <40 pg/ml (GDF11 low, cutoff determined following receiver operating characteristic (ROC) curve analysis of combined ELISA results) presented around 70% of aplastic bone marrows at early response assessment (Day 15) following induction chemotherapy, the subgroup of patients presenting GDF1140 pg/mL (GDF11 high) presented around 61% of blastic bone marrows following induction therapy (FIG. 5A). Therefore, GDF11 high AML bear features associated with reduced responses to induction chemotherapy regimen.

[0227] We next analyzed the number of chemotherapy courses necessary to achieve complete remission (CR) in GDF11 low and GDF11 high subgroups. We observed that around 69% of patients of the GDF11 low subgroup achieved CR following 1 course of intensive chemotherapy regimen whereas 72% of patients in the GDF11 high group received two or more courses of chemotherapy to achieve CR. Therefore, increased circulating GDF11 levels segregated with impaired chemotherapy responses in AML patients. Thus, increased GDF11 levels at diagnosis are associated with impaired remission induction.

[0228] Neutralization of GDF11 Induces Cell Apoptosis in AML Cell Lines.

[0229] We searched for the cellular consequences of GDF11 neutralization in AML cells. AML cells (MOLM-14, THP1, HL60 and MV4-11 cell lines) were cultured with growing doses of GDF11 blocking antibodies and apoptosis was evaluated by Annexin V staining. All AML cells were sensitive to anti-GDF11 blocking antibodies suggesting that GDF11 expression in required for AML cell growth (FIG. 6A). In addition, anti-GDF11 antibodies also induce a cell growth arrest (FIG. 6B). Further use of different commercial antibodies confirmed previous data showing data blocking antibodies could induce cell apoptosis (FIG. 6C).

[0230] Neutralization of GDF11 Induces Cell Apoptosis in Primary AML Blasts.

[0231] Primary AML blasts from eight different patients were cultured with growing doses of GDF11 blocking antibodies and apoptosis was evaluated (FIG. 7). Among the eight samples, only one sample was insensitive to anti-GDF11 blocking antibodies (subject 6), samples corresponding to subjects 5 and 6 showed intermediate sensitivity to GDF11 blockade (around 50% apoptosis) whereas samples corresponding to subjects 1,3,4,7 and 8 were highly sensitive to GDF11 inhibition.

CONCLUSION

[0232] Primary refractoriness to induction chemotherapy therapy regimen occurs in 10-25% of newly diagnosed acute myeloid leukemia patients (AML) and is associated with dismal prognosis.sup.4. Here we show that increased GDF11 serum levels in intermediate cytogenetic risk AML is associated with impaired responses to chemotherapy regimen. GDF11 levels do not segregate with the most frequent somatic mutations found in the disease therefore defining a new patient subgroup with possible consequence to disease management. Indeed GDF11 appeared as a new marker predicting responses to chemotherapy. Therefore, determination of GDF11 levels at diagnosis could be a new indicative factor which could help to define groups for alternative therapies (e.g. targeted therapy) instead of serial chemotherapy regimen. In addition, increased GDF11 in patients with favorable prognosis (e.g. CBF, CEBPa, NPM1 mutations) could also be an indication for allogenic BM transplantation. This could reduce toxicity of chemotherapy in patients which do not fit with this therapy and therefore ameliorate patients' survival by reducing undesired toxicity associated with this therapy.

[0233] Our studies identify GDF11 as a new soluble factor involved in an autocrine loop controlling chemotherapy resistance in AML. We show that overexpression of GDF11 is correlated with impaired induction chemotherapy responses and in patients with impaired responses to consolidation therapy. Molecular mechanisms involved are still unknown and are currently under investigation in our laboratory. The fact that GDF11 overexpression is not associated with commonly found somatic mutations in NK-AML suggests that GDF11 expression is independent on specific alteration of AML cell biology. Further studies will be necessary to clarify this point but reactive oxygen species (ROS) could be one of the candidates implicated in GDF11 overexpression since we have previously show that ROS induces GDF11 expression and ROS are involved in AML biology.sup.15,19. Hence, we propose that GDF11 functions as new regulator of chemotherapy resistance and probably though the modulation of ROS levels which impacts in AML biology. Although our study focused on leukemia, our data imply that the GDF11 overexpression could be involved in chemotherapy resistance of other malignant hematological diseases. We also demonstrated that GDF11 inhibition with blocking antibodies induces apoptosis both in AML cell lines and in primary AML blasts. These preliminary data open major perspectives in the therapy of AML patients.

TABLE-US-00001 TABLE1 Usefulnucleotideandaminoacidsequencesforpracticingtheinvention SEQID NO Nucleotideoraminoacidsequence 1 MVLAAPLLLGFLLLALELRPRGEAAEGPAAAAAAAAAAAA (GDF11 AGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIK AA SQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQ sequence) GDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPL CCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRL KPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDF KQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEG LHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTV DFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQ QANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS 2 tccccgccccccagtcctccctcccctcccctccagcatggtgctcgcggccccgctgctgctgggcttc (GDF11 ctgctcctcgccctggagctgcggccccggggggaggcggccgagggccccgcggcggcg nucleic gcggcggcggcggcggcggcggcagcggcgggggtcgggggggagcgctccagccggcca acid gccccgtccgtggcgcccgagccggacggctgccccgtgtgcgtttggcggcagcacagc sequence) cgcgagctgcgcctagagagcatcaagtcgcagatcttgagcaaactgcggctcaaggag gcgcccaacatcagccgcgaggtggtgaagcagctgctgcccaaggcgccgccgctgcag cagatcctggacctacacgacttccagggcgacgcgctgcagcccgaggacttcctggag gaggacgagtaccacgccaccaccgagaccgtcattagcatggcccaggagacggaccca gcagtacagacagatggcagccctctctgctgccattttcacttcagccccaaggtgatgttcacaaagg tactgaaggcccagctgtgggtgtacctacggcctgtaccccgcccagccacagtctacctgcagatctt gcgactaaaacccctaactggggaagggaccgcagggggagggggcggaggccggcgtca catccgtatccgctcactgaagattgagctgcactcacgctcaggccattggcagagcatcgacttcaag caagtgctacacagctggttccgccagccacagagcaactggggcatcgagatcaacgcctttgatccca gtggcacagacctggctgtcacctccctggggccgggagccgaggggctgcatccattcatggagcttcg agtcctagagaacacaaaacgttcccggcggaacctgggtctggactgcgacgagcactcaagcgagtcc cgctgctgccgatatcccctcacagtggactttgaggctttcggctgggactggatcatcgcacctaagc gctacaaggccaactactgctccggccagtgcgagtacatgttcatgcaaaaatatccgcatacccattt ggtgcagcaggccaatccaagaggctctgctgggccctgttgtacccccaccaagatgtccccaatcaac atgctctacttcaatgacaagcagcagattatctacggcaagatccctggcatggtggtggatcgctgtg gctgctcttaaggtgggggatagaggatgcctcccccacagaccctaccccaagacccctagccctgccc ccatccccccaagccctagagctccctccactcttcccgcgaacatcacaccgttccccgaccaagccgt gtgcaatacaacagagggaggcaggtgggaattgagggtgaggggtttgggggaaagggg aagcaggggcatagtcagggtggggagtgtttgaagtttgcagatgagaaggtttgacaaaaagacagag agatgtagagacagtgatagagacagaggaacaaaaagagcagcagtgagaaggcaaaga gagaggcagaagagacagacgaggcagagacaaaacactgagaaagagactgaaatggag taataaatgaaagccccacaccaagcctcctttcttccactggcaaggtgaggggcttggtatagtttgg ggagatcccctgactattcagtaggagaagaaatcaaaaatccattcttttctccttctctccctccaac agtggccaggggaaggggaagtgagggcaggggcaaaaagatttgggaatttttatttatttatttattg tgacttttcatttttttggtatttggctttactggaataggagggcccctgcccactgtgccccgtttat cccttattccccaaaccctgctctccccaacacctactcacttaagcacttgtataaagcctccagggtt gggaatgggagtaaagggcaagagggcggacacatgaagtttagtttctaacccatcatcaccctaactc aaccttttctgagccaaatggcttgaattgaagccagttgtcatggaaatagtaagaggttagggtttaa gagctggggatgcgggggtgggagagagaaccctcaacatccaggatctatataatgagagctactttaa accctcaggtccaccctcatgatgctgagttatttagccagagggtgcagcctgcttatgcccaaattcc ctcagccaagagagagaccaaagagcctctggaatggccctgctcccagcctctatcttcaggtcaatta gagagagtatagagaccccagagtcccctgggtctggaaagcgttaggagaggtcaagaa aggagcagtaaggaggctgaaggttacagggcatttgaatccaaatcactgctctgggctagggaataga gccagcagaccaaggtgggaaggattctggaagggggacattttagtctcctaaccccaaagctcagggt ggaagaggggagaacaaggaagcagagtgtataattattttttccttttatttttggaatctaacagtac ctggcagcagggaggggaaagtacagtggggaaaagcatctgacaaggccagttagaaca gaggatgggaaggatggagactcccgggcttggaaggctaggaagcaggcagagactggt tgccatttcaagtcactagctaggcccattcattcctcccacaaccctgacccattctcctctggactca ctgtgcctcagtttcttcccctcaatggaatgagaaatgacagcacccgccacagccaagagatgaattc tgagcacttaccacgggcactttatggacataaaatacctctcgctgtgggacagataaccagggcacca gagtagtggtgaagagatgtgaggcttaagaggagtcacaggcttcagagtacaagttcccctctgcctc ccagctggacagtgcctagaagccaaggagttgagaatctcctgatccacaccctatccttacttcacca ccaggcctcttggctccaggcaagagcttagaggatgtcaggagaggtgggggtaagaatcttcagcaaa actgtcactctaagtagagccagcagttacgggtctgataaaaacagtactgaactaaagtaaagcccaa gctggtgagcaaaactggatggctcattcttcccaagagcatgactctcccccttggccagttggtggaa ggggcaaaggtatgtgaccacccttgagaaggtgatgttggtgagctttaacatcttattcctattctta tagtgagaaagtgaaacaagatctttcagtagaggaatgggcagggctgttaggctcttcagcttgcctt cacccatatagcagctatgctaaccccaagcctctctggccctgttcttcatccttccttctgccccaat cctgaaggacaagacacacccggccatcaacaccactcacatttccttggtggaaggaaaggaacagaga agtgaagaacagatacctccctccaaggtcaaatgcctcgtgatcttggcagagtagggattgggcaata agcatcaggtatcttccctctacagattctagagagctggggcattaaatatgggggacacttagaatac agctccttaaataccaccaaataaagacctttgtgtgtgtgtggtgggtggggggggggcaggggtcttt ctcttatgaacataaatctgtgagctgaagtctcattcccctgttcctccctacccccaaagaggcacag agtgaagggacttggggggcacagctcagcaacccagtgggagttagcaccccctcccaccttatgatgt gtgtggacctggccagtgcccctctgaacatatcattattagtgtaattatcatttattttgtgtatttg tcacattgtgtgcatgacagcctttgttaagggtgtctgaggagtatggagctgacaggggcattggaat gccaggaaagaacttcttcaactgagatcaaggcttcctggagggaaccactgcaaaaaggccatcaggc agttttcaagttatgtgacagagggcaaagacggccatagggtgctctgagttttgggatggtcacatga cacaatccagcacttgaacctgaaaaaaaaaataaaagcggtcaaagagtttagaattca 3 MGAAAKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRTNQTGVE (human PCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKD ActRIIA SPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLV precursor PLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQL polypeptide) LEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGM KHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNEL CHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACI ADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRID MYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVV HKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERI TQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL 4 MTAPWVALALLWGSLWPGSGRGEAETRECIYYNANWELERTNQSGLE (human RCEGEQDKRLHCYASWX(AorR)NSSGTIELVKKGCWLDDFNCYDRQEC ActRIIB VATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVL precursor AYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKP polypeptide) LQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTP GMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWN ELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDL TAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAF LRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQE VVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEE RVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

REFERENCES

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