Glycosphingolipid biosynthesis inhibitors for treatment of tumors dependent on oncogenes GOLPH3 and GOLPH3 cluster oncogenes

Abstract

The object of the present invention is the use of glycosphingolipid biosynthesis inhibitors for the treatment of tumors that are dependent on the oncogene GOLPH3 as well as on two oncogenes similar to GOLPH3 (GOLPH3-L and LCS, which form a GOLPH3 cluster with GOLPH3). Another object of the invention is the use of GOLPH3 genomic amplification detection as a predictive marker for identifying tumors responsive to said inhibitor.

Description

[0001] The present invention aims to utilize glycosphingolipid biosynthesis inhibitors for the treatment of tumors dependent on the oncogene GOLPH3 and on two GOLPH3-like oncogenes (GOLPH3-L and LCS, which together with GOLPH3 form the GOLPH3 cluster). Another object of the invention is the use of Golph3 as a marker for identifying tumors sensitive to the inhibitors. Existing technology

[0002] Golgi phosphoprotein 3 (GOLPH3) is an oncoprotein encoded by a gene found in a region of the human genome that is frequently amplified in various solid tumors, including melanoma, lung cancer, breast cancer, glioma, and colorectal cancer (Rizzo R et al., Tissue Cell 2017;49(2 Pt A):170-174). The reported frequency varies depending on the source (database or publication). For example, according to an authoritative and conservative database, the amplification frequency of GOLPH3 is 11% in squamous cell lung cancer, 7% in lung adenocarcinoma, and 6% in uterine cancer (The Cancer Genome Atlas).

[0003] GOLPH3 is not only amplified on the genome, but also overexpressed (in a large number of tumors). The overexpression is associated with poor prognosis in a variety of tumors, including 52% of breast cancer and 41% to 53% of glioblastoma (Sechi S et al., Int. J. Mol. Sci. 2020; 21: 933). The percentages also vary depending on the source (database or publication). Finally, the oncogene GOLPH3 leads to chemotherapy resistance, which is a serious obstacle to tumor drug treatment.

[0004] The oncogenic mechanism of GOLPH3 (particularly regarding its ability to induce chemotherapy resistance) is believed to be due to the ability of the oncogene to selectively stimulate the trafficking of growth receptors to the plasma membrane, thereby activating the receptors (Farber-Katz SE et al., Cell. 2014 156(3):413-27; Buschman MD et al., Cancer Res. 2015 75(4):624-7). Consistent with this finding, other authors subsequently demonstrated that GOLPH3 acts by activating signaling pathways, including those initiated by growth factor receptors (such as the EGF receptor) or those mediated by oncogenic kinases and GTPases (such as PI3K kinase, AKT, mTOR, RAS, etc.). (Zhi-Ping Zhou et al., Int J Oncol. 2018 53(3):1183-1192; Ming-Zhen Wang Molecular Medicine Reports 2018 17: 542-548).

[0005] Recently, Rizzo R et al. proposed in EMBO J. 2021: 15;40(8) that the carcinogenic effect of GOLPH3 is related to glycosphingolipid (GSL) metabolism. The authors demonstrated that GOLPH3 acts by increasing the levels and therefore the activity of a group of GSL synthases, which are (in order of action): lactosylceramide synthase (LCS), GB3 synthase (GB3S), GM3 synthase (GM3S) and GD3 synthase (GD3S), thereby increasing the amount of their metabolites (LC, GB3, GM3, GD3) and their precursors glucosylceramide (GC) and ceramide (Cer) (see Figure 1 schematic diagram).

[0006] The metabolites are biologically active; ceramide is a tumor suppressor that inhibits growth and induces apoptosis; whereas GB3 and GD3 activate SRC and growth factor receptors, which stimulate cell growth. Therefore, the result of reduced ceramide and increased GB3 and GD3 leads to inhibition of apoptosis and enhanced stimulation of proliferation. This action can protect tumor cells from the effects of stress-induced ceramide production (see below) and stimulate cell proliferation, which may promote tumor development. The effects of other oncogenes belonging to the GOLPH3 cluster, GOLPH3-L and LCS, are very similar to those induced by GOLPH3 ( Figure 1 The metabolic pathway of GSL and the mechanism of action of GOLPH3 are as follows. Figure 1 shown.

[0007] A series of synthesis reactions (shown in horizontal sequence) starting from serine (SER) and palmitic acid (PALM) generate sphingosine (SP), which is then used by ceramide synthase (CerS) to form Cer. Cer (black squares) is a tumor suppressor and functionally a key molecule in the system because its formation induces apoptosis, while its depletion leads to cell proliferation. In the Golgi complex, Cer is consumed by glucosylceramide synthase (GCS, the drug target, in the concentric circles) and converted to glucosylceramide (GC). GC is then transferred from the cytoplasmic leaflet to the luminal leaflet of the Golgi membrane by the "flippase" P-glycoprotein (P-gp, which is also one of the known molecular "pumps" that can squeeze various molecules, including drugs, out of the cell).

[0008] In the lumen, GC is subsequently converted to lactosylceramide (LacCer) by LCS. LacCer is a substrate for the synthesis of all GSL complexes, including GB3 and globosides, GM3 and gangliosides, and LC3 and lactosides. The mechanism of action of the GOLPH3 cluster is shown in the central vertical rectangle. GOLPH3 (grey background) is a regulator of GSL metabolism. It acts on the enzymes mentioned in the text (arrows), increasing their levels and thereby increasing their activity. This increase has two complementary effects: a) depleting Cer, thereby attenuating apoptosis, and b) increasing the formation of enzyme products that stimulate SRC and growth factor receptors (RTKs), i.e., stimulating cell proliferation. Regarding other oncogenes belonging to the GOLPH3 cluster, GOLPH3-L (grey background, top) is very similar to GOLPH3 and has the same effects, while LCS (grey background, top) is the primary mediator of GOLPH3's actions and has the same effects as GOLPH3.

[0009] Furukawa et al. in Cancer Sci. (2019; 110:1544-1551) and Ogretmen B in Nature Reviews (2018; 18:33-50) described the role of GSLs as markers and cell signaling regulators in tumors. The possibility of using inhibitors of glucosylceramide synthase (GCS) to inhibit GSL biosynthesis to counteract tumor growth has been studied for some time; for example, Inokuchi et al. in Cancer Letters (38, 1987, 23-30) described the antitumor activity of 1-phenyl-2-decanoylamino-3-morpholinopropanol (PDMD), a UDP-glucosylceramide glucosyltransferase inhibitor, in a mouse tumor model (Ehrlich ascites).

[0010] Stefanovic et al. reported in Oncotarget, 2016; 7(7):8254-8263 that some GSL biosynthesis inhibitors, especially GCS inhibitors (PDMD, imipramine), enhance the activity of sorafenib against hepatocellular carcinoma cells in vitro and in vivo. Tyler et al., Experimental Cell Research, 2015; 336: 23-32, described the enhancement of cisplatin cytotoxicity against melanoma cells and pleural mesothelioma by PDMD. The potential antitumor activity of GCS inhibitors belonging to the iminosugar chemical class (such as miglustat and its derivatives) in Lovo and HCT116 cells and mouse colorectal adenoma models is described in Int. J. Mol. Sci. 2021, 22, 10539; or those belonging to the ceramide analog class, such as PDMP and eliglustat; or P-gp inhibitors, such as zosuquidar and its analogs (Gottesman MM et al., FEBS Lett. 2006 13;580(4):998-1009) or such as the tamoxifen metabolite 4-hydroxytamoxifen (Morad SAF et al., Biochim Biophys Acta. 2015; 1851(9): 1134–1145.).

[0011] However, despite some positive data, demonstrating the efficacy of GSL metabolic inhibitors as anti-tumor drugs in cell and animal models has so far remained rare and unpredictable. Most researchers have published data on experimental conditions and cell lines in which they can demonstrate the effects of GSL metabolic inhibition, but have not published data on cases in which such inhibitors are ineffective, a common occurrence in research.

[0012] It has now been found that by comparing the effects of GSL metabolic inhibitors on tumor cells characterized by amplification of GOLPH3 and genes belonging to the GOLPH3 cluster with the effects of the inhibitors on tumor cells that do not exhibit such amplification, only a limited proportion of tumor cell lines, namely tumor cell lines characterized by GOLPH3 amplification (for example, 10% of squamous cell lung cancer cell lines as mentioned above) or tumor cell lines characterized by significant overexpression of genes belonging to the GOLPH3 cluster, have reproducible and predictable sensitivity to GSL metabolic inhibitors in cell growth assays and stress resistance assays (for example, DNA damage caused by chemotherapy).

[0013] This is consistent with the results reported in the DEPMAP database (The Cancer Dependency Map Program at the Broad Institute), which showed that GCS depletion significantly inhibited the growth of two of the 100 tumor cell lines grown in vitro adhering to a cell matrix. The success rate is far from reaching the standard of clinical utility. The anti-tumor effect of such inhibitors has never been confirmed in clinical practice. Only the efficacy of miglustat and its derivatives in reducing osteoclast activation and bone destruction associated with multiple myeloma has been reported (J Clin Invest. 2015;125(6):2279–2292). However, the effect is limited to osteoclasts.

[0014] Overall, these data suggest that only a subset of tumors characterized by GOLPH3 and GOLPH3 cluster amplification are sensitive to the effects of GSL biosynthesis inhibitors and that glycosphingolipid synthesis inhibitors hold promise for pharmacological application in the clinical setting, a common scenario in the development of many targeted drugs.

[0015] It should be noted that, prior to the present study, it would not have been possible to predict that inhibitors of GSL metabolism would be systemically and predictably active only in GOLPH3-amplified tumor cells. In fact, glycosphingolipid metabolism has the potential to activate mitogenic signaling in all mammalian cells. The reason that only GOLPH3-amplified tumors respond may be that Golph3 is a potent activator of this metabolic pathway because it can simultaneously activate different enzymes of this metabolic pathway (see Figure 1 ), and therefore has been used as a defense mechanism by various types of tumors.

[0016] Description of the Invention

[0017] It has been found that GSL biosynthesis inhibitors ( Figure 1 Schematic diagram) can be used selectively to treat tumors that rely on GOLPH3 cluster oncogenes. Specifically, the study found that, when treated with the inhibitors, GSL biosynthesis inhibitors only inhibited the growth of tumor cell lines that: 1) express at least one oncogene belonging to the GOLPH3 cluster; and 2) are grown under stress conditions caused by the presence of chemotherapeutic drugs or loss of cell-matrix anchorage. In contrast, in the absence of stress, under matrix-adherent growth conditions, the inhibitors had little effect on the growth of the same tumor cell lines.

[0018] These observations suggest that: 1) the presence of oncogenes belonging to the GOLPH3 cluster can serve as predictive markers to identify tumors sensitive to inhibition of GSL metabolism; 2) the inhibitor works only in these tumor cell lines under stress conditions, abolishing their chemotherapy resistance and resistance to loss of anchorage to the matrix (which normally induces apoptosis). Both of these situations have considerable clinical significance. Chemotherapy resistance is one of the major issues involved in cancer treatment, and resistance to loss of anchorage is known to reflect the ability of tumors to grow and metastasize in vivo.

[0019] GCS inhibitors, already used in clinical practice to treat other diseases (hereditary disorders involving lysosome accumulation), have been used as GSL metabolism inhibitors, as have P-GP inhibitors. Therefore, this strategy involves repositioning GSL metabolism inhibitors for anti-tumor use, thereby eliminating expensive development steps.

[0020] Tumors targeted by the GOLPH3 cluster oncogenes that could be beneficially treated with GSL metabolism inhibitors include liver, lung, prostate, ovarian, and colon cancers, as well as melanoma.

[0021] Examples of GSL metabolism inhibitors include iminosugars (such as miglustat), ceramide analogs (such as iliglustat or its tartrate, venglustat or N-[(1R,2R)-2-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-hydroxy-1-(1-pyrrolidinylmethyl)ethyl]-nonanamide (GENZ-123346), P-GP (a flippase essential for GSL metabolism) inhibitors such as zoquinoline and tamoxifen derivatives, which are metabolites thereof. Miglustat and iliglustat are drugs already in use, while venglustat is currently undergoing first-level clinical trials. These compounds are collectively referred to as GSL metabolism inhibitors.

[0022] The table below lists other compounds known to have inhibitory activity against human glucosylceramide synthase (GCS) and compounds known to have inhibitory activity against P-GP and therefore inhibit GSL metabolism that have been used in scientific experiments and evaluations. The right column cites one of the many references in the scientific literature.

[0023]

[0024] Figure 2A1 、 2A2 and 2B present data by way of examples to support the above conclusions.

[0025] The GSL metabolic inhibitors listed above were used in tumor cell lines expressing oncogenes belonging to the GOLPH3 cluster. The results showed that these inhibitors could suppress the oncogene-induced resistance to apoptosis (caused by loss of anchorage to the matrix) and resistance to chemotherapy drugs (in this case, etoposide).

[0026] The growth of tumor cell lines was evaluated under different experimental conditions. Twelve tumor cell lines were used, divided into six pairs, each derived from six different tumor types. Each pair consisted of a tumor cell line expressing an oncogene belonging to the GOLPH3 cluster and a tumor cell line from the same tumor type that did not express the oncogene. The growth of the cell lines was observed under three experimental conditions: 1) not anchored to a matrix; 2) exposed to chemotherapeutic drugs; and 3) undisturbed growth while adhered to a matrix.

[0027] Figure 2A1Figure 3: Growth of prostate, colon, and liver tumor cell lines expressing oncogenes belonging to the GOLPH3 cluster was inhibited by miglustat under anchorage-independent conditions (at concentrations ranging from 0 to 50 micromolar, similar to concentrations known to inhibit GCS enzymes in cells) (top panel, light gray), whereas growth of tumor cell lines from the same tumor type but not expressing GOLPH3 was not inhibited by miglustat under the same conditions (middle panel, dark gray). Growth of cell lines expressing oncogenes belonging to the GOLPH3 cluster under matrix-adherent and stress-free conditions (bottom panel, black) was not inhibited by miglustat. Inhibition of GSL synthesis was approximately 70% (50 micromolar miglustat). Depletion of GOLPH3 cluster members reached 90% by 25 nanomolar siRNAs targeting oncogenes belonging to the GOLPH3 cluster expressed in the cell lines and inhibited growth more significantly than miglustat at these concentrations (not shown).

[0028] Figure 2A2 Shows the Figure 2A1 The same results were seen, but for melanoma, lung cancer, and ovarian cancer cell lines.

[0029] Figure 2B The results show the growth of tumor cell lines adhered to the matrix in the presence of chemotherapeutic drugs (chemoresistant). A cell line that does not express an oncogene belonging to the GOLPH3 cluster (LnCap) exhibits cell cycle arrest (significantly increased p21 synthesis) after treatment with the chemotherapeutic drug etoposide, which is not altered by co-treatment with etoposide. In contrast, a cell line expressing an oncogene belonging to the GOLPH3 cluster (DU145) is resistant to etoposide but loses this resistance in the presence of iliglustat. Treatment with iliglustat alone is ineffective. Similar results were achieved with the P-GP inhibitor zoquinoline.

[0030] Method Description

[0031] Reagents

[0032] Cell lines and culture conditions:

[0033] Ovarian cancer NIHOVCAR3 and TOV21G, liver cancer HepG2 and Hep3B, skin melanoma A375 and SKMEL2, colon cancer HCT116 and RKO, prostate cancer DU145 and LnCaP, and lung cancer A549 and PC14 cell lines were purchased from the American Tissue Collection (ATTC, USA). DU145, A375, A549, HCT116, and HepG2 cell lines were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, and streptomycin; SKMEL5, Hep3B, and RKO cell lines were cultured in MEM supplemented with 10% FBS, 1 mM non-essential amino acids (NAA), 2 mM L-glutamine, 100 U / ml penicillin, and streptomycin; and NIHOVCAR3, TOV21G, LnCaP, and PC14 cell lines were cultured in RPMI supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, and streptomycin. All tumor cell lines used in this study were tested and confirmed to be mycoplasma-free and cultured at a controlled temperature (37°C) and atmosphere (5% CO2 and 95% air).

[0034] Drugs: Miglustat (from Cayman), Eliglustat (from Cayman), Genz-123346 (from Sigma), Etoposide (from Sigma), Azoquinoline (from SelleckChem).

[0035] Small RNA interference (RNAi):

[0036]

[0037] Primary and secondary antibodies:

[0038]

[0039] Treatment method

[0040] Gene silencing via siRNA

[0041] The siRNAs used in this study were obtained from Sigma-Aldrich (Table 1). siRNA transfection was performed using a lipofectamine lipofection kit or RNAi Max (Thermofisher Scientific, USA) at concentrations ranging from 5 to 50 nM, as previously described (Rizzo et al., 2021). Silencing efficiency was assessed by Western blot or qPCR using specific primers.

[0042] Blocking GSL synthesis by inhibitors

[0043] GCS enzyme inhibitors, such as miglustat, iliglustat, and N-[(1R,2R)-2-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-hydroxy-1-(1-pyrrolidinylmethyl)ethyl]-nonanamide (Genz-123346), or P-gp inhibitors such as oxaquindal, were used at two or three different concentrations depending on the dosage type to block glycosphingolipid biosynthesis in various tumor cells. In the growth assay, new medium supplemented with the drug was added every three days.

[0044] Chemotherapy drug treatment

[0045] Treat cells with etoposide at concentrations ranging from 10 to 40 µM for 12-16 hours.

[0046] test

[0047] Cell growth assay

[0048] 1. Growth in the absence of anchorage (anchorage-independent)

[0049] 2. Growth of isolated cell clones (clonogenic analysis)

[0050] 3. Growth under adhesion conditions (Under adhesion conditions).

[0051] Both anchorage-independent and clonogenic growth assays were performed as previously described by Rizzo et al., EMBOJ, 2021. Growth and cell viability assays under adherent conditions were performed by measuring XTT or ATP according to the manufacturer's protocol.

[0052] Cell apoptosis assay

[0053] The cleavage of PARP by caspase-3 was assessed by immunoblotting as an indicator of apoptosis activation.

[0054] Cell cycle arrest assay

[0055] The p21 level was measured by immunoblotting as an index of cell cycle arrest.

[0056] Glycosphingolipid metabolism

[0057] For the analysis of glycosphingolipid metabolites, a method based on radiolabeling of cellular sphingosine was used, followed by extraction of metabolites and separation by thin-layer chromatography (TLC) as previously described by Rizzo et al., EMOJ, 2021.

[0058] Immunoblotting

[0059] Proteins were labeled with the primary antibodies mentioned in Table 2 and visualized with goat anti-rabbit or goat anti-mouse secondary antibodies IgG-HRP (Santa Cruz, USA) diluted 1:10,000.

[0060] statistics

[0061] Error bars correspond to the standard deviation (SD) or standard error of the mean (SEM) indicated in the figure legends. Statistical analysis was performed using the Student's t test, with ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (ns, not significant).

[0062] The mechanism behind the conclusions

[0063] Growth in the presence of chemotherapy drugs (chemoresistant)

[0064] Chemotherapeutic drugs act by damaging the DNA of target cells. This triggers an apoptotic response, which prevents cells with severely damaged DNA from continuing to proliferate in the body. This response is mediated by multiple mechanisms, including activation of p53 (the most extensively characterized) and increased ceramide, which leads to cell cycle arrest, essential for DNA repair. If the damage is excessive, apoptosis can occur unless its effects are counteracted by oncogenes that directly promote survival and growth or by mechanisms that rapidly deplete ceramide. GOLPH3 and oncogenes belonging to the GOLPH3 cluster activate both of these protective mechanisms.

[0065] In addition, an important regulatory mechanism activates GOLPH3 specifically to counteract stress conditions; DNA damage and increased ceramide activate a mechanism based on PKD and DNAPK kinases, which phosphorylate GOLPH3 and strongly enhance the action of GOLPH3, thereby consuming ceramide and promoting the production of growth GSLs. Under physiological conditions, this mechanism acts as a control system to reduce the increase in ceramide and the apoptotic effects caused by DNA damage, which, if not alleviated, may be uncontrolled and destructive in non-tumor cells. However, in tumor cells that overexpress GOLPH3, this system induces excessive suppression of apoptotic signals and excessive increase in growth signals, which together lead to chemotherapy resistance and increased tumor proliferation, making the tumor cells "invulnerable" to the apoptotic response induced by chemotherapy drugs, such as Figure 3 The mechanism shown.

[0066] The above regulatory mechanism specifically explains why the oncogene GOLPH3 selectively and effectively protects tumor cells from apoptosis under stress conditions (such as DNA damage, in which large amounts of ceramide are produced), that is, because the oncogene represents a potent chemotherapy resistance factor, but is not very active in the absence of stress. Therefore, this also explains why GCS inhibitors such as iliglustat (which block the consumption of ceramide and the synthesis of growth-promoting GSLs) can eliminate the chemotherapy resistance caused by the influence of GOLPH3 on GSLs and its oncogenic effect.

[0067] Growing without anchoring

[0068] GOLPH3 also plays a protective role against apoptosis under other stress conditions, such as loss of anchorage to the matrix, in which significant ceramide production occurs. Although less characterized, the mechanisms of apoptosis caused by loss of anchorage under these conditions and the mechanisms of apoptosis resistance mediated by GOLPH3 are similar to the mechanisms of chemotherapy resistance described above. Similar conclusions apply to other oncogenes in the GOLPH3 cluster.

[0069] Figure 3 The schematic diagram in the figure shows a regulatory mechanism based on PKD and DNAPK, which enhances the ability of GOLPH3 to induce resistance to DNA damage and loss of anchoring to the matrix, as well as the role of GCS inhibitors as inhibitors of the properties of GOLPH3. DNA damage (top rectangle, gray background, left) induces a significant increase in ceramide synthase transcription, thereby increasing the production of ceramide. Some ceramide is used to generate sphingomyelin (SM) and diacylglycerol (DAG), which in turn activates PKD kinase (top rectangle, gray background, right). The kinase phosphorylates GOLPH3 and enhances its activity. The second effect of DNA damage is to directly activate DNAPK kinase (top rectangle, gray background, right), which acts similarly to PKD and further enhances the effect of GOLPH3. Therefore, GOLPH3 is specifically enhanced in events involving DNA damage (or loss of anchoring, followed by ceramide production). This makes the effect of GOLPH3 particularly effective under the above-mentioned severe stress conditions.

[0070] GCS inhibitors (such as iliglustat) or P-GP inhibitors (such as zoquinoline) can block the consumption of ceramide by GCS and subsequent enzymes and the synthesis of growth-promoting GSLs, thereby blocking the anti-apoptotic and growth-promoting effects of GOLPH3.

[0071] For the therapeutic uses contemplated by the present invention, GCS or P-GP inhibitors can be formulated into compositions suitable for oral or parenteral administration using conventional techniques. Dosage depends on the type and stage of the disease, as well as the patient's weight, sex, and age, and can be determined based on preclinical and clinical trials. For miglustat, eliglilustat, and other inhibitors already in clinical use, oral dosages are generally consistent with those for known, approved therapeutic indications.

[0072] The activity of GCS and P-GP inhibitors in inhibiting the proliferation of tumor cells dependent on GOLPH3 cluster oncogenes was demonstrated using lung, ovarian, prostate, skin (melanoma) and colon cancer cell lines.

Claims

1. A glycosphingolipid metabolism inhibitor for use in the treatment of tumors that are dependent on the expression of GOLPH3 and GOLPH3 cluster oncogenes and resistant to chemotherapy.

2. The glycosphingolipid metabolism inhibitor for use according to claim 1, which is an iminosugar.

3. The glycosphingolipid metabolism inhibitor for use according to claim 2, wherein the iminosugar is miglustat.

4. The glycosphingolipid metabolism inhibitor for use according to claim 1, which is selected from N-[(1R,2R)-2-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-hydroxy-1-(1-pyrrolidinyl-methyl)ethyl]-nonanamide, iligliucoma, iligliucoma tartrate, vengliucoma and zoquinoline.

5. The glycosphingolipid metabolism inhibitor for use according to any one of claims 1 to 4, wherein the GOLPH3 and GOLPH3 cluster oncogene expression-dependent and chemotherapy-resistant tumors are liver cancer, lung cancer, prostate cancer, ovarian cancer, colon cancer, and melanoma.

6. A method for classifying a tumor as sensitive or resistant to treatment with a glycosphingolipid inhibitor comprising measuring the expression levels of GOLPH3 and GOLPH3 clusters.

7. The method of claim 6, wherein the tumor is resistant to chemotherapy.

8. Use of GOLPH3 genomic amplification detection as a predictive marker for identifying tumors responsive to glycosphingolipid metabolism inhibitors.

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