COMPOSITIONS AND METHODS RELATED TO LATROPHILINS AS BIOMARKERS FOR HAEMATOPOIETIC CELL CANCER

Abstract

Disclosed is the use of latrophilin expression as a biomarker for the diagnosis of haematopoietic cell cancer in a subject, together with methods for diagnosis and a kit for the detection of latrophilin expression on white blood cells collected from a subject.

Claims

1-28. (canceled)

29. A kit for the detection of one or more latrophilin isoforms on white blood cells collected from a subject, the kit comprising: i) a latrophilin capture agent; ii) one or more factors to enhance latrophilin expression; iii) one or more latrophilin ligand; and iv) means to visualise products of exocytosis from the white blood cells.

30. The kit according to claim 29, wherein the latrophilin capture agent is an antibody against latrophilin.

31. The kit according to claim 30, wherein the antibody against latrophilin is specific for latrophilin 1, latrophilin 2 or latrophilin 3, or any combination thereof.

32. The kit according to claim 29, wherein the kit further comprises one or more reagent to visualise captured cells.

33. The kit according to claim 32, wherein the one or more reagent to visualise captured cells is an antibody against a cell-surface protein or a cell detection reagent.

34. The kit according to claim 29, wherein the latrophilin capture agent is coated on at least one surface.

35. The kit according to claim 34, wherein the at least one surface is an internal surface of a well in a microtitre plate.

36. The kit according to claim 29, wherein the one or more factor to enhance latrophilin expression is a pro-inflammatory factor.

37. The kit according to claim 29, wherein the one or more factor to enhance latrophilin expression is selected from a lipopolysaccharide (LPS), stem cell factor (SCF) or anti-Tim-3 antibody.

38. The kit according to claim 37, wherein the lipopolysaccharide is derived from Pseudomonas aeruginosa.

39. The kit according to claim 29, wherein the one or more latrophilin ligand induces exocytosis.

40. The kit according to claim 29, wherein the one or more latrophilin ligand is selected from: a latrophilin antibody, alpha-latrotoxin and Lasso/teneurin-2.

41. The kit according to claim 29, wherein the means to visualise products of exocytosis from the white blood cells include one or more of an antibody, or a radio- or fluorophore-labelled ligand.

42-49. (canceled)

50. The kit according to claim 29, wherein the kit further comprises instructions to monitor the effectiveness of a therapy to treat or slow the progression of a haematopoietic cell cancer, or to decide on initiation, continuation or discontinuation (ending) of the therapy.

51. The kit according to claim 29, wherein the kit further comprises instructions to characterise a stage or status of a haematopoietic cell cancer.

52. The kit according to claim 51, wherein, the haematopoietic cell cancer is of myeloid origin.

53. The kit according to claim 51, wherein the haematopoietic cell cancer is leukaemia.

54. The kit according to claim 50, wherein the therapy is chemotherapy, radiotherapy, bone marrow and/or stem cell transplant and/or one or more agent to stimulate white blood cell and/or stem cell production in the body, steroids and new chemical, biochemical or biological entities.

55. (canceled)

Description

[0084] The present invention will now be described in more detail with reference to the following figures in which:

[0085] FIG. 1 shows the effects of LPS and alpha-latrotoxin (LTX) on LPHN1/2 expression, IL-6 exocytosis and mTOR activity in U937 human AML cells. U937 cells were exposed to 1 μg/ml LPS, 500 pM LTX or a combination of these two ligands for 24 h. Following cell lysis, latrophilin protein levels were analysed by Western blot. IL-6 release was measured by ELISA (A). Phospho-S2448 mTOR and phospho-T389 p70 S6K1 were detected using ELISA and Western blot respectively (B). Western blot images show a representative experiment of six, which gave similar results. Data are mean values±SD (n=6). *-p<0.05; **-p<0.01; ***-p<0.001.

[0086] FIG. 2 shows the effects of LPS and LTX on LPHN1/2 expression, IL-6 exocytosis and mTOR activity in THP-1 human AML cells. THP-1 cells were exposed to 1 μg/ml LPS, 500 pM LTX or a combination of these two ligands for 24 h. Following cell lysis, latrophilin protein levels were analysed by Western blot. IL-6 release was measured by ELISA (A). Phospho-S2448 mTOR and phospho-T389 p70 S6K1 were detected using ELISA and Western blot respectively (B). Western blot images show a is representative experiment of six which gave similar results. Data are mean values±SD (n=6). *-p<0.05; **-p<0.01; ***-p<0.001.

[0087] FIG. 3 shows that expression of LPHN1/2 proteins in human AML cell lines is an mTOR-dependent process. U937 (A) and THP-1 (B) cells were exposed to 1 μg/ml LPS for 4 h with or without 1 h pre-treatment with 10 μM rapamycin. Latrophilin protein levels were analysed by Western blot. Western blot images show a representative experiment of six which gave similar results. Data are mean values±SD (n=6). *-p<0.05; **-p<0.01.

[0088] FIG. 4 shows the effects of LPS and LTX on IL-6 exocytosis and mTOR activity in primary human AML cells. Primary human AML-PB001F cells were exposed to 1 μg/ml LPS, 500 pM LTX or a combination of these two ligands for 24 h. Following cell lysis, IL-6 release was measured by ELISA. Phospho-S2448 mTOR levels in cell lysates were also characterised using ELISA. Data are mean values±SD (n=6). *-p<0.05; **-p<0.01.

[0089] FIG. 5 shows that primary human AML cells, but not healthy primary human leukocytes, express functional LPHN 1 and LPHN 2. Primary AML-PB001F cells were exposed for 4 h (A) and healthy human leukocytes for 4 h (B) or 24 h (C) to indicated concentrations of LPS, SCF and anti-Tim-3. Following cell lysis, latrophilin protein levels were analysed by Western blot. Phospho-S2448 mTOR was measured by ELISA. Furthermore, non-treated THP-1 cells, primary human AML cells and primary healthy human leukocytes, as well as those stimulated for 24 h with 1 μg/ml LPS were subjected to quantitative real time polymerase chain reaction qRT-PCR (D). Images show one representative result of at least four experiments, which gave similar results. Data are mean values±SD (n=4). *-p<0.05; **-p<0.01.

[0090] FIG. 6 shows the expression of LPHN3 in THP-1 and U-937 ML cell lines in the absence and presence of either (1) peptidoglycan (PGN), which is a ligand of Toll-like receptor 2 (TLR2), or (2) stem cell factor (SCF). Cells were exposed to the indicated concentrations of stimuli for 4 h, followed by Western blot analysis of LPHN3 expression. Images are from one experiment representative of five independent experiments all of which gave similar results. Quantitative data are the mean values±SEM (n=5).

[0091] FIG. 7 is a Western Blot showing the expression of LPHN1 in primary human chronic lymphocytic leukaemia cells (PC, positive control; rat brain extract). Images are from one experiment representative of three independent experiments which is gave similar results.

[0092] FIG. 8 illustrates the mRNA levels of LPHNs 1, 2 and 3 expressed in primary human chronic lymphocytic leukaemia mononuclear cells (CLL-BM001M, provided by AllCells) isolated from the bone marrow of a patient with chronic lymphocytic leukaemia. A, Agarose gel electrophoresis of amplified gene fragments, after 50 PCR cycles. Arrows indicate the expected positions of specifically amplified fragments. Bottom bands, unused oligonucleotide primers. Amplification in the absence of cDNA was used as a negative control. The β-actin gene was used as a house-keeping gene for quantitative analysis of relative gene expression. B, Quantitative analysis of the expression of respective genes. The data are shown as mean values±SEM from three independent qRT-PCR experiments (n=3), normalised to the expression of β-actin. Only mRNA of LPHN1 was clearly identified, while LPHN2 and LPHN3 mRNAs were undetectable.

[0093] FIG. 9 is a Western Blot showing the expression of LPHNs 1 and 3 in a human cell line derived from malignant mast cells. Images are from one experiment representative of three independent experiments which gave similar results.

EXAMPLE 1

[0094] The following example demonstrates that latrophilin (LPHN) isoforms 1 and 2 are expressed in human acute myeloid leukaemia (AML) cell lines (U937 and THP-1) in an mTOR-dependent manner and are capable of enhancing IL-6 exocytosis.

Materials and Methods

[0095] RPMI-1640 medium, foetal calf serum and supplements were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained from Nunc (Roskilde, Denmark) as well as from Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies to mTOR and β-actin, as well as rabbit polyclonal antibodies against phospho-52448 mTOR, were obtained from Abcam (Cambridge, UK). Antibodies against phospho-T389 and total p70 S6 kinase 1 (p70 S6K1) were obtained from Cell Signalling Technology (Danvers, Mass. USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from Li-Cor (Lincoln, Nebr. USA). ELISA-based assay kits for the detection of IL-6 were purchased from R&D Systems (Abingdon, UK). LTX was isolated from the venom of black widow spider Latrodectus sp. as described by Ashton A. C. et al (Biochimie (2000) 82 453-468). All other chemicals were of the highest grade of purity and commercially available.

[0096] THP-1 and U937 human leukaemia monocytic macrophages were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in RPMI 1640 media supplemented with 10% foetal calf serum, penicillin (50 IU/ml) and streptomycin sulphate (50 μg/ml).

[0097] Levels of LPHN 1 and LPHN 2, total and phospho-T389 p70 S6K1 were determined by Western blot analysis and compared to β-actin in order to determine equal protein loading, as described by Yasinska I. M. et al (Cell. Mol. Life Sci. (2014) 71 699-710). Li-Cor goat secondary antibodies, conjugated with fluorescent dyes, were used according to the manufacturer's protocol to visualise the proteins of interest using a Li-Cor Odyssey imaging system. Western blot data were analysed quantitatively using Odyssey software and values were normalised against respective β-actin bands.

[0098] Phosphorylation of mTOR was monitored using ELISA assays as recently described (Yasinska I. M. et al (supra); Abooali M. et al (2014) Sci. Rep. 4 6307; Prokhorov A. et al (supra)). Briefly, ELISA plates were coated with mouse anti-mTOR antibodies and blocked with 2% BSA. Cell lysates were then added to the wells and kept at room temperature for at least 2 h (under constant agitation). After extensive washing with TBST buffer (Tris-Buffered Saline and Tween 20), anti-phospho-52448 mTOR antibody was added and plates were incubated for at least 2 h at room temperature with constant agitation. Plates were then washed with TBST buffer and incubated with 1:1000 Horse Radish Peroxidase-labelled goat anti-rabbit IgG in TBST buffer. After extensive washing with TBST, bound secondary antibodies were then detected by the peroxidase reaction (ortho-phenylenediamine/H.sub.2O.sub.2).

[0099] Concentrations of IL-6 released into the cell culture media were analysed by ELISA (R&D Systems assay kit) according to the manufacturers protocol.

[0100] Each experiment was performed at least three times and statistical analysis was conducted using a two-tailed Student's t test or, where appropriate, a one-way ANOVA with post-hoc Tukey test, with correction for multiple pairwise comparisons.

Results

[0101] Expression of LPHN 1 and LPHN 2 in human AML cell lines—U937 and THP-1—was investigated. Cells were stimulated for 24 h with 1 μg/ml LPS, 500 pM LTX or a combination of these ligands. LPS is a pathogen-associated molecular pattern shared by Gram-negative bacteria and is recognised by the Toll-like receptor 4 (TLR4), which is highly expressed by human myeloid cells. Importantly, LPS was chosen because TLR4 may be targeted by endogenous ligands (like proteins released by dysfunctional mitochondria) to promote expression/release of IL-6 and other important cytokines/growth factors required for leukaemia cell survival.

[0102] It was found that both U937 and THP-1 cells expressed detectable levels of LPHN 1 and LPHN 2 (FIG. 1A and FIG. 2A). Commercially available mouse brain extract was used as a positive control. In both U937 and THP-1 cells, LPHN1/2 expression levels were significantly up-regulated by LPS but not by LTX (FIG. 1A and FIG. 2A). However, the IL-6 exocytosis induced by LPS was highly up-regulated by LTX in both U937 and THP-1 human AML cells (FIG. 1A and FIG. 2A).

[0103] It was also found that LPS, but not LTX, significantly activated the mTOR pathway, increasing its activating phosphorylation at position S2448 and also the phosphorylation of its substrate, p70 S6 kinase 1 (p70 S6K1) at position T389. This was clearly observed in both cell lines (FIG. 1B and FIG. 2B).

[0104] Since mTOR is a master regulator of myeloid cell translation, the role of the mTOR pathways in LPS-induced up-regulation of LPHN1/2 protein levels was investigated.

[0105] Both U937 and THP-1 cells were exposed to 1 μg/ml LPS for 4 h with or without 1 h pre-treatment with 10 μM rapamycin (a highly specific mTOR inhibitor). The results show that, unlike 24 h stimulation, 4 h exposure to LPS led to a moderate increase in LPHN1/2 expression in both U937 and THP-1 cells. However, the effect was stronger in THP-1 cells. Rapamycin attenuated the expression of LPHN 1 and LPHN 2 in both cell lines (FIG. 3A and B). This suggests that latrophilin protein accumulation strongly depends on the mTOR pathway which displays a clear background activity in resting cells and is further increased upon stimulation (for example LPS, FIGS. 1B and 2B).

Conclusion

[0106] The results demonstrate that resting AML cells from the U937 and THP-1 human cell lines express both LPHN 1 and LPHN 2. The expression was up-regulated by LPS via the mTOR pathway (a master regulator of myeloid cell translational pathways). LTX, a specific high affinity latrophilin ligand which causes an increase in cytosolic calcium and massive exocytosis in neurons, significantly enhanced LPS-induced exocytosis of IL-6 in both cell lines. Since the production of IL-6 protein depends on mTOR activation, and activity of the mTOR pathway was not up-regulated by LTX in these cell lines, it is clear that LTX was up-regulating IL-6 exocytosis and not its biosynthesis.

[0107] Interestingly, both LPHN 1 and LPHN 2 protein levels were nearly abolished when the cells were pre-treated with the mTOR inhibitor rapamycin before exposure of both U937 and THP-1 cells to LPS. This observation suggests that the process of LPHN1/2 expression strongly depends on the mTOR pathway. As demonstrated in the FIGS. 1B and 2B, in both cell lines there is a background activity of the mTOR pathway which is further up-regulated by LPS.

EXAMPLE 2

[0108] The following example demonstrates that functional LPHN 1 and LPHN 2 are expressed in primary human AML cells but not in primary healthy human leukocytes.

Materials and Methods

[0109] Unless indicated otherwise, materials and methods were identical to those used in Example 1.

[0110] Primary human AML mononuclear cells (AML-PB001 F, newly diagnosed/untreated) were purchased from AllCells (Alameda, Calif., USA) and handled in accordance with manufacturer's instructions.

[0111] Primary human leukocytes were obtained from buffy coat blood (prepared from healthy donors) purchased from the National Health Blood and Transfusion Service (NHSBT, UK) following ethical approval (REC reference: 12/WM/0319). Mononuclear-rich leukocytes were obtained by Ficoll-density centrifugation according to the manufacturer's protocol. Cell numbers were determined using a haemocytometer and diluted accordingly with HEPES-buffered Tyrode's solution before treatment as indicated.

[0112] Human Stem Cell Factor (SCF) protein was produced in E. coli and purified in accordance with published protocols (Wang C. et al (2008) Appl. Biochem. Biotechnol. 144 181-189).

[0113] To obtain anti-TIM3 mouse monoclonal antibodies, human TIM-3 Ig-like V-type domain (extracellular domain, position 22-124) was produced in E. coli, refolded from inclusion bodies and purified according to standard protocols. Monomer and aggregates of the protein were collected and used to immunise 8 week old C56BL/6 mice (5 for protein monomer and 5 for aggregates) using standard protocols for mouse immunisation. Spleens from highly responsive mice were collected and used to create hybridomas. Antibodies were screened according to their affinity to the Tim-3 monomer (via ELISA and SPR). The highest affinity monoclonal antibody was selected and used in this study (Prokhorov A. et al (supra)).

Results

[0114] Experiments were carried out to ascertain whether or not LPHN 1 and LPHN 2 are expressed and function in primary human AML cells. AML-PB001 F primary human mononuclear blasts were used and exposed for 24 h to 1 μg/ml LPS, 500 pM LTX or a combination of these ligands.

[0115] It was found that LPS up-regulated both mTOR activation and IL-6 release by these cells. LTX alone did not influence these processes but, in combination with LPS, significantly up-regulated both mTOR activation and IL-6 exocytosis (FIG. 4).

[0116] To analyse LPHN1/2 expression levels in AML-PB001F cells, the cells were exposed to mTOR activators (1 μg/ml LPS, 0.1 μg/ml SCF or 2 μg/ml anti-Tim-3 antibody) for 4 h. It was found that each stimulus significantly increased phosphorylation of S2448 in mTOR and therefore mTOR activation (FIG. 5A). SCF induced the strongest effect due to a high level of Kit (SCF receptor) expressed by these cells. All stimuli also significantly up-regulated LPHN1/2 expression as detected by Western blot analysis.

[0117] LPS and SCF significantly up-regulated LPHN2 expression in AML-PB001 F primary cells, while LPHN2 expression was non-significantly up-regulated by anti-Tim-3 (FIG. 5A). This observation is in line with results reported by others on the intensity of mTOR-dependent effects provoked by LPS, SCF and anti-Tim-3 in human AML cells (Prokhorov, A. et al (supra)).

[0118] Quantitative real-time PCR (RT-PCR) experiments suggested that THP-1 cells and primary AML cells, but not healthy primary human leukocytes, produce LPHN1 mRNA (FIG. 5D). Furthermore, 1 μg/ml LPS was able to upregulate LPHN1 mRNA levels slightly in THP-1 cells. However, no significant changes were observed after 24 h exposure. Intriguingly, 24 h stimulation of primary AML cells with 1 μg/ml LPS led to a significant increase in LPHN1 mRNA levels. In primary healthy human leukocytes even 24 h exposure to 1 μg/ml did not induce LPHN1 mRNA expression (FIG. 5D).

Conclusion

[0119] These experiments demonstrate that similar effects may be observed in primary human AML cells, using primary AML-PB001F mononuclear blasts obtained from leukaemia patients, to the effects seen in AML cell lines. It has been found that resting primary human AML cells and those exposed to mTOR stimuli (LPS, SCF and anti-Tim-3) express both LPHN1 and LPHN2. mTOR activators were able to up-regulate the levels of both proteins in primary AML cells, as also seen in cell lines. LTX increased LPS-induced IL-6 release.

[0120] However, LTX also up-regulated LPS-induced mTOR-activating phosphorylation in AML cells, while it failed to do so in the cell lines. This is likely to be a result of low expression of TLR4 (LPS receptor) in AML cells, as demonstrated by the provider of the AML cells. In these cells, LPS binding to TLR4 causes some mTOR activation via phosphorylation of its S2448, but a large proportion of mTOR molecules remains unphosphorylated/inactive and any additional activation of the mTOR signalling mechanisms may still be able to increase mTOR activation. In cell lines, where TLR4 expression is much higher, LPS-induced mTOR activation may be approaching its maximum and LTX may not be able to increase mTOR activation further.

[0121] Importantly, primary leucocytes obtained from healthy donors (buffy coats) did not express any latrophilin. Furthermore, this expression was not inducible by 4 or 24 h exposure of the cells to any of the stimuli which showed positive effects in AML cells (LPS, SCF or anti-Tim-3). This means that expression of functional LPHN1 and LPHN2 is specific only to leukaemia cells.

[0122] An important observation was also made in quantitative RT-PCR experiments, which have demonstrated that healthy primary human leukocytes do not produce LPHN1 mRNA. This means that a basic protein expression profile change is taking place in leukocytes during malignant transformation. The LPHN1 gene appears to be repressed in leukocytes. However, LPHN1 expression does take place in malignantly transformed cells. The value of this protein to malignant leucocytes is obvious—these are weak cells that require production of certain growth factors (like VEGF) and cytokines (including IL-6) to survive and progress the disease. However, leucocyte exocytosis is an energy-consuming process associated with major cytoskeleton alterations. This could be beyond the capabilities of malignant cells. Therefore, they are likely to require powerful mechanisms inducing exocytosis, like those available in neurons.

EXAMPLE 3

[0123] The following example demonstrates LPHN3 protein expression in human acute myeloid leukaemia cells.

Materials and Methods

[0124] THP-1 human myeloid leukaemia monocytes and U937 human myeloid monocytes were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in RPMI 1640 media supplemented with 10% foetal bovine serum, penicillin (50 IU/ml) and streptomycin sulphate (50 μg/ml).

[0125] THP-1 and U-937 cells were exposed for 4 h to indicated concentrations of peptidoglycan (PGN, Sigma Aldrich, UK), or stem cells factor (SCF, Human SCF was a kind gift of Dr. Luca Varani, IRB, Bellinzona, Switzerland). Non-treated cells incubated under similar conditions were used as a control. Cells were then harvested, lysed (whole cell extracts were prepared using 50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet-40, 1 mM PMSF buffer) and subjected to Western blot analysis of LPHN3.

[0126] The levels of the LPHN3 isoform was analysed using Western blot. The polyclonal rabbit anti-peptide antibodies PAL1, PAL2 and PAL3 against LPHN1, 2 and 3 respectively have been produced in-house and previously described (Davydov et al (2009) Bull. Exp. Biol. Med. 148: 869-873; Silva et al (2011) Proc. Natl. Acad. Sci. U.S.A. 108: 12113-12118). LPHN3 was determined as described in (Gonsalves Silva et al (2015) Oncotarget 6: 33823-33833; Prokhorov et al (2015) Int. J. Biochem. Cell Biol. 59: 11-20). Fluorescently labelled antibodies (Li-Cor) were used according to the manufacturer's protocol to visualise the proteins of interest using an Odyssey imaging system (Li-Cor). Western blot data were subjected to quantitative analysis using the Odyssey software and values were normalised against respective β-actin bands.

Results

[0127] As can be seen in FIG. 6, LPHN3 was expressed in both THP-1 and U-937 human myeloid leukaemia cell lines in a statistically significant amount. THP-1 and U-937 cells express different relative amounts of LPHN3 and LPHN1.

Conclusion

[0128] These results show that LPHN3 is expressed in human myeloid leukaemia cell lines and support a role for this receptor isotype in leukaemia.

EXAMPLE 4

[0129] In this example, the expression of LPHN isoforms in primary chronic lymphoid leukaemia cells was investigated.

Materials and Methods

[0130] Primary human bone marrow-derived CLL mononuclear cells (CLL-BM001F, newly diagnosed/untreated leukaemia) were obtained from AlICells (Alameda, Calif., USA) and handled in accordance with manufacturer's protocol. Cells were analysed following ethical approval (REC reference: 16-SS-033).

[0131] The primary malignant mononuclear cells were extracted using a lysis buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet-40, 1 mM PMSF buffer) and subjected to Western blot analysis of LPHN1, 2 and 3.

[0132] The levels of LPHN1, 2 and 3 isoforms were analysed using Western blot. The polyclonal rabbit anti-peptide antibodies PAL1, PAL2 and PAL3 against LPHN1, 2 and 3 respectively have been produced in-house and previously described (Davydov et al (supra); Silva et al (supra). Expression levels of LPHNs 1-3 were determined as described in Gonsalves Silva et al (supra); Prokhorov et al (supra). Fluorescently labelled antibodies (Li-Cor) were used according to the manufacturer's protocol to visualise the proteins of interest using an Odyssey imaging system (Li-Cor). Western blot data were subjected to quantitative analysis using the Odyssey software and values were normalised against respective β-actin bands.

[0133] A separate fraction of cells was used for quantitative real-time PCR (qRT-PCR) to analyse mRNA levels of LPHN isoforms. Total RNA was extracted using the Illustra RNAspin Midi RNA isolation kit (GE Healthcare) and quantified spectroscopically using Nanodrop 2000® (Thermo Scientific). cDNA was then synthesised with the help of Transcriptor First Strand cDNA Synthesis Kit (Roche), which was used in accordance with the manufacturer's protocol. Relative quantification of LPHN mRNAs was performed using SYBR Green I Master reaction mix (Roche) and a LightCycler 480 (Roche). The house-keeping gene β-actin was used as a reference gene in all cases.

[0134] The following primers were used at a final concentration of 0.5 μM:

TABLE-US-00001 LPHN1: (SEQ ID NO: 1) 5′-AGCCGCCCCGAGGCCGGAACCTA-3′; (SEQ ID NO: 2) 5′-AGGTTGGCCCCGCTGGCATAGAGGGAGTC-3′; LPHN2: (SEQ ID NO: 3) 5′-CACAACGTCGACCTCACACTACCAGTCAAGCCTG-3′; (SEQ ID NO: 4) 5′-TGGCACTATTAGAGACTAGTCACCAGCTGCATTTG-3′; LPHN3: (SEQ ID NO: 5) 5′-GACCTGGGCCTTTGGACTCATGTA-3′; (SEQ ID NO: 6) 5′-CGCCGCTGGCAATGCTGTA-3′; Actin: (SEQ ID NO: 7) 5′-TTCGCGGGCGACGATGC-3′; (SEQ ID NO: 8) 5′-GGGGCCACACGCAGCTCATT-3′.

[0135] PCR reactions were begun with incubation at 95° C. for 3 min 30 s, then proceeded for 45 cycles of 95° C. for 10 s, 60° C. for 20 s and 72° C. for 10 s. Fluorescence levels were detected at 80° C. in each cycle. A final elongation step took place at 72° C. for 5 min. Raw fluorescence data were analysed using LinRegPCR quantitative PCR data analysis programme (Ruijter et al (2009) Nucleic Acids Res. 37: e45). Amplified products were validated using 1.5% agarose gel containing ethidium bromide.

Results

[0136] As shown in the Western Blot in FIG. 7, only LPHN1 protein was detectable in CLL cells. Similar results were obtained using qRT-PCR in which only the mRNA of LPHN1 was detectable (data not shown). mRNA for LPHNs 2 and 3 was not seen in CLL cells.

[0137] FIG. 8 shows the mRNA levels of LPHNs1, 2 and 3 in primary human chronic lymphocytic leukaemia (CLL) cells in which only LPHN1, but not LPHN2 or LPHN 3 is expressed. This is seen on both mRNA and protein levels.

Conclusion

[0138] The expression of LPHN1 in CLL cells suggests a possible role for this latrophilin isoform in lymphatic leukaemias.

EXAMPLE 5

[0139] In this example, the expression of LPHN isoforms in LAD2 human mast cells derived from malignant mast cells originally isolated from a mast cell sarcoma was investigated. Mast cells and myeloid hematopoietic cells are derived from the same type of stem cells (human myeloid progenitor cells). Therefore, both mast cells and myeloid leukocytes are considered to be “myeloid haematopoietic cells”. Thus both myeloid leukaemia and mast cell malignancies are present in myeloid cell cancers.

Materials and Methods

[0140] LAD2 mast cells were kindly provided by A. Kirshenbaum and D. Metcalfe (NIH, USA) (Kirshenbaum et al (2003) Leuk. Res. 27: 677-682). Cells were cultured in Stem-Pro-34 serum-free media supplemented with 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml) and recombinant human SCF (100 ng/ml).

[0141] Non-treated LAD2 human malignant mast cells and those exposed for 24 h to 0.1 μg/ml human IgE were lysed (whole cell extract was prepared using 50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet-40, 1 mM PMSF buffer) and subjected to Western blot analysis as described in Example 4.

Results

[0142] As shown in FIG. 9, LPHNs 1 and 3 were detectable in malignant mast cells, while LPHN2 was not detectable. Interestingly, the levels of LPHN1 and LPHN3 were decreased in IgE-treated cells. IgE binds to high-affinity IgE receptors (FccRI) on mast cells and plays a role in their activation during type-I hypersensitivity reactions (allergies) and in certain autoimmune diseases. Actin staining was employed to confirm equal protein loading in each well of the gel.

Conclusion

[0143] LAD2 human malignant mast cells express LPHN1 and LPHN3 proteins, while LPHN2 was not detectable by Western blot analysis.

[0144] The results presented here clearly demonstrate that functional LPHN1 and LPHN2 are expressed in human AML cells but not in healthy leucocytes. Furthermore, in healthy leucocytes, LPHN1/2 expression is not inducible. In AML cells, latrophilin is likely to contribute to exocytosis of growth and angiogenic factors required for proliferation of AML cells and bone marrow angiogenesis. LPHN3 is expressed in human myeloid leukaemia cell lines while LPHN1, but not the two other known isoforms, is detectable in human CLL cells. Human malignant mast cells (another type of myeloid haematopoietic cell cancer) express LPHN1 and LPHN3, but not LPHN2.

[0145] Therefore, expression of latrophilin isoforms should be suitable as a novel biomarker for haematopoietic cell cancer diagnostics such as leukaemia and provide novel targets for therapy and drug delivery.