CSE inhibitors for use in tumor treatment in combination with MAPK inhibitors

Abstract

The present invention relates to cystathionine-γ-lyase (CSE) inhibitors for use in combination with (one or more) MAPK inhibitors, particularly inhibitors of BRAF V600 variants, for the treatment of cancer, preferably tumors, in patients, and preferably for the prevention or delay of the onset or development of resistance in patients to treatment of said cancer with MAPK inhibitors, particularly inhibitors of BRAF V600 variants. The present invention also relates to combination therapies, optionally accompanied by diagnosis, and pharmaceutical compositions, combinations, and kits for such therapies.

Description

[Technical Field]

[0001] The present invention relates to cystathionine-γ-lyase (CSE) inhibitors for use in combination with (one or more) MAPK inhibitors, particularly BRAF V600 variant inhibitors, for use in the treatment of cancer, preferably a patient's tumor, particularly melanoma, preferably for use in preventing or delaying the onset or development of resistance in the patient to treatment of said cancer with MAPK inhibitors. The present invention also relates to combination therapies, optionally accompanied by diagnosis, as well as pharmaceutical compositions, combinations, and kits for such therapies. [Background technology]

[0002] Cutaneous melanoma is the most deadly form of skin cancer. Early diagnosis increases the chances of survival, but the overall prognosis is poor due to the rapid progression of the disease and the frequent occurrence of distant metastases. The most common oncogenic mutations that drive tumorigenesis occur in the BRAF, NRAS, and NF1 genes. Approximately 50% of patients with cutaneous melanoma have the V600E activating mutation in the serine / threonine protein kinase B-raf (Braf) oncogene, which leads to overactivation of the MAPK / ERK pathway and therefore to uncontrolled cancer cell proliferation (Davies et al., 2002). Vemurafenib (V therapy), the first FDA-approved targeted therapy for BrafV600E mutant melanoma, has a high response rate, but unfortunately, tumors rapidly acquire resistance (Sosman et al., 2012). Combination inhibition of V600E variant Braf with dabrafenib and the downstream bispecific mitogen-activated protein kinase kinase 1 / 2 (MEK1 / 2, also known as MAP2K1 / 2) and trametinib (DT-treated) extends progression-free survival and overall survival, but resistance to this combination therapy is almost unavoidable (Manzano et al., 2016; Prahallad et al., 2012; Robert et al., 2015; Sosman et al., 2012; Sun et al., 2014).

[0003] It is increasingly recognized that treatment resistance in various cancers is associated with extensive reprogramming of metabolic pathways in cancer cells (Luis et al., 2020; Yoo and Han, 2022). The ability of melanoma cells to rapidly proliferate is supported by increased aerobic glycolysis (Hall et al., 2013), which is promoted by BRAFV600E mutagenic activation of the Braf / MEK / ERK pathway (Haq et al., 2013). On the other hand, BrafV600E inhibition induces major metabolic changes in melanoma cells. This includes a shift from aerobic glycolysis to increased mitochondrial respiration (Corazao-Rozas et al., 2016), which induces the production of reactive oxygen species (ROS) and leads to a change in the redox environment (Khamari et al., 2018; Wang et al., 2018). The purpose of this study was to clarify the mechanistic aspects that may link this increase in ROS production to the development of resistance to dabrafenib / trametinib (DT) and vemurafenib (V) therapy.

[0004] The inventors have found that a trans-sulfurization pathway specifically designed for the production of cysteine ​​from methionine plays a major role in this process. Apart from their standard function in cysteine ​​production, the trans-sulfases cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) play crucial roles in the biosynthesis of small signaling molecules, including hydrogen sulfide and cysteine ​​persulfide (Ida et al., 2014; Kumar and Banerjee, 2021). In recent years, the important functions of these reactive sulfur species (RSS) in human physiology and pathology have become clear, making them a focus of redox biomedical research (Cirino et al., 2023; Cortese-Krott et al., 2017; Wallace and Wang, 2015; Wang et al., 2021). In particular, their carcinogenic functions are increasingly recognized (Coletta et al., 2012; Czikora et al., 2022; Erdelyi et al., 2021; Pavlova et al., 2022; Szabo, 2016).

[0005] With most targeted therapies that block driver oncogenes (such as BRAF V600E), cancer cells can develop acquired resistance with repeated administration (Sosman et al., 2012). Acquired resistance to treatment remains an unresolved problem in cancer, such as melanoma, and solutions that can overcome or prevent such resistance are crucial for further advances in the treatment of melanoma and other tumors.

[0006] European Patent Publication No. 3563870 discloses a PD-1 axially linked antagonist for use in the treatment or delaying progression of BRAF antagonist-resistant melanoma, the PD-1 axially linked antagonist being administered in combination with a MEK inhibitor. However, this combination therapy does not prevent the development of resistance to BRAF inhibitors.

[0007] International Publication No. 2015004636 discloses a method for treating melanoma, comprising administering a CDK (cyclin-dependent kinase) inhibitor and a BRAF inhibitor and / or MEK inhibitor to a subject in need of treatment. The melanoma being treated may be resistant BRAF mutant melanoma. However, this method does not prevent the development of resistance to BRAF / MEK inhibitors.

[0008] International Publication No. 2015161230 discloses a method for treating cancer in an individual, comprising administering a MAPK inhibitor and one or more AXL inhibitors, Met inhibitors, and PI3K inhibitors. In one embodiment, the present invention relates to a method for reducing resistance to a MAPK inhibitor, comprising administering a MAPK inhibitor and an AXL inhibitor. Cancer may be carcinoma, sarcoma, leukemia, breast cancer, melanoma, lung cancer, etc.

[0009] International Publication No. 2021171260 discloses a triple drug combination comprising dabrafenib, an ERK inhibitor, and a RAF inhibitor or PD-1 inhibitor for use in the treatment of cancer, such as breast cancer, melanoma, or non-small cell lung cancer. The triple combination is particularly useful for the treatment of colorectal cancer (including advanced or metastatic colorectal cancer) that is a BRAF gain-of-function variant or a BRAF V600E / D / K variant. The ERK inhibitor can target multiple resistance mechanisms to BRAF and MEK inhibitors, and thus can avoid resistance.

[0010] International Publication No. 2022259157 discloses a triple drug combination comprising dabrafenib, trametinib, and an SHP2 inhibitor for the treatment of cancers, such as breast cancer, melanoma, or non-small cell lung cancer. The SHP2 inhibitor is (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridine-4-yl)thio)pyrazine-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine. The triple combination is particularly useful for the treatment of colorectal cancer (including advanced or metastatic colorectal cancer) that is a BRAF gain-of-function variant or a BRAF V600E variant. The triple combination has the potential to uniquely target intrinsic and acquired resistance mechanisms in cancer cells driven by BRAF V600.

[0011] Today, combinations of vemurafenib and cobimetinib, dabrafenib and trametinib, or encorafenib and binimetinib are the most widely used combination targeted therapies in patients with melanoma who have the V600 mutation (based on Giunta et al., 2020).

[0012] Jiang et al. (Jiang et al., 2023) showed that AOAA enhances the antitumor (colorectal cancer) effect of regorafenib. However, the authors noted that AOAA was used to inhibit PSAT1, and since AOAA is not sufficiently specific, a more specific PSAT1 inhibitor is needed. This paper confirmed that AOAA acts via PSAT1 and affects cellular metabolism in several ways. It is also important to note that AOAA is not a selective CSE inhibitor, as is evident from Jiang et al. Therefore, AOAA in the context of Jiang et al. cannot be considered a CSE inhibitor, and those skilled in the art could not derive different guidance from Jiang et al. Furthermore, we found that treatment-naive BRAF V600 mutant melanomas were not sensitive to AOAA even at concentrations 10 to 20 times higher than those used by Jiang et al.

[0013] Sun et al. (Sun et al., 2016) suggested that PAGs with CSEi activity enhance the anticancer effect of sorafenib in hepatocellular carcinoma (HCC), and also showed that the underlying mechanism of this phenomenon is mediated by the metallothionein-1G (MT-1G) protein. The results allowed for the conclusion that sorafenib induces MT-1G, which is important for the development of acquired resistance to sorafenib in HCC cells via inhibition of sorafenib-induced cell death (ferroptosis). Furthermore, it was shown that PAGs inhibit metallothionein synthesis, and that PAG treatment results in reduced levels of MT-1G.

[0014] The canonical and non-canonical functions of CSE inhibited by PAG were not investigated at all.

[0015] Furthermore, while sorafenib may inhibit the proliferative mutant protein in melanoma cells, it is not specific to Braf V600E, which also inhibits the wild-type Braf protein that performs essential physiological functions, and its mechanism of action is fundamentally different from that of specific BRAF V600 inhibitors. In addition, sorafenib also targets several other signaling proteins, including VEGFR, PDGFR, c-Kit, and RET, which play important roles in regulating cell proliferation and angiogenesis. Moreover, sorafenib has also been shown to inhibit the cystine-glutamate countertransporter involved in cystine uptake, which is crucial for cellular cysteine ​​supply. Thus, a study published by Sun et al. used sorafenib in combination with an inhibitor of CSE, which is responsible for cysteine ​​production in the event of extracellular restriction or inhibition of its uptake, to inhibit cysteine ​​uptake. As a result, Sun et al. did not mention the inhibition of resistance that could develop into targeted therapy, given the fundamentally different mechanisms of action of the two drugs.

[0016] Therefore, the beneficial effects of CSE inhibition in resistant CSEs cannot be inferred with respect to Braf V600 / MAPK inhibitors.

[0017] Liu et al. (Liu et al., 2021) showed that I194496 inhibits the growth of triple-negative breast cancer (TNBC).

[0018] Wang et al. (Wang et al. 2019) described that compound I157172 inhibits metastasis in breast cancer.

[0019] Li et al. (Li et al., 2021) summarized the progress made in the control of breast cancer by using inhibitors against H2S-producing enzymes, and showed that CSE inhibitors I194496, I157172, PAG, BCA, AVG have anti-cancer activity.

[0020] Liu et al (Liu et al., 2021), Wang et al. (Wang et al., 2019) and Li et al. (Li et al., 2021) reported the anti-cancer effects of CSE inhibition in some tumors, but none of these publications described the anti-tumor effects of CSE inhibitors in either BRAF V600 mutant tumors or melanomas.

[0021] The inventors found that inhibition of CSE has no anti-tumor effect in treatment-naive BRAF V600 mutant melanoma or in combination with BRAF V600E / MAPK inhibitors.

[0022] However, surprisingly, it was found that inhibition of CSE delays the onset of acquired resistance to BRAF V600E and MAPK inhibitors.

[0023] In the prior art, progress has been made in finding treatments for resistant cancers or preventing the development of resistance to anti-cancer agents. However, solutions that can prevent or at least delay the onset of resistance in cancer treatment are still needed.

[0024] The present invention provides a novel solution for treating cancer, in particular by preventing and / or delaying the onset of resistance to known cancer treatments. This solution is based on the discovery of the molecular mechanisms underlying the onset of resistance to MAPK inhibitors (in particular BRAF inhibitors and / or MEK inhibitors). SUMMARY OF THE INVENTION

[0025] The prior art does not describe the use of a CSE inhibitor in combination with a MAPK inhibitor in BRAF V600-mutant positive cancer for preventing or delaying the onset or development of acquired resistance in patients to the treatment of BRAF V600-mutant positive cancer.

[0026] The inventors have surprisingly found that inhibition of CSE is beneficial for preventing resistance induced by MAPK inhibitor treatment in BRAF V600-mutant positive cancer, while the CSE inhibitor itself has no anti-tumor effect in treatment-naive BRAF V600-mutant melanoma or in combination with a BRAF V600E / MAPK inhibitor.

[0027] 1. The present invention relates to a cystathionine-γ-lyase (CSE) inhibitor for use in combination with one or more MAPK inhibitors, particularly MAPK inhibitors of the BRAF V600 mutant, for treating BRAF V600-mutant positive cancer in a patient, preferably a BRAF V600-mutant positive tumor, and preferably for preventing or delaying the onset or development of resistance in the patient to the treatment of said cancer with a MAPK inhibitor. Preferably, the MAPK inhibitor comprises a BRAF V600 inhibitor, particularly a specific inhibitor of the BRAF V600 mutant. More preferably, the mutation is selected from the group consisting of V600D, V600K, V600R or V600E, more preferably V600K or V600E; more preferably V600E. Preferably, the MAPK inhibitor comprises a BRAF inhibitor, particularly an inhibitor of the BRAF V600 mutant, and / or a MEK inhibitor. The present invention also relates to cystathionine-γ-lyase (CSE) inhibitors for use in preventing or delaying the onset or development of resistance to cancer treatment with one or more MAPK inhibitors in patients treated with one or more MAPK inhibitors. The present invention also relates to a cystathionine-γ-lyase (CSE) inhibitor for use in the treatment of a patient who has been treated with one or more MAPK inhibitors, the patient being resistant to or at risk of developing resistance to the one or more MAPK inhibitors. The present invention also relates to a combination of a cystathionine-γ-lyase (CSE) inhibitor and one or more MAPK inhibitors for use in the treatment of a patient's cancer to prevent or delay the onset or development of resistance to the one or more MAPK inhibitors. The term "one or more MAPK inhibitors" can be replaced with "MAPK inhibitors." Resistance to such one or more MAPK inhibitors should preferably be understood as resistance to at least one of such one or more MAPK inhibitors, and includes resistance to each of such one or more MAPK inhibitors. Preferably, cancer is a tumor. Preferably, the patient is a mammalian patient, preferably a human patient. In certain embodiments, the BRAF inhibitor is different from sorafenib. In certain embodiments, the CSE inhibitor is different from aminooxyacetic acid (AOAA).

[0028] 2. In preferred embodiments, cancer, preferably tumors, include melanoma, skin cancer, epithelial cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, thyroid cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, gastrointestinal neuroendocrine tumors, neuroblastoma, glioma, astrocytoma, leukemia, hairy cell leukemia, hepatobiliary cancer, nephroblastoma (Wilms' tumor), histiocytosis, Langerhans cell histiocytosis and Erdheim-Chester disease, preferably melanoma, colorectal cancer, colon cancer, rectal cancer, lung cancer; and in particular melanoma, selected from the group consisting of these, CSE inhibitors for use according to paragraph 1.

[0029] 3. In preferred embodiments, the MAPK inhibitor is a BRAF V600 variant inhibitor and / or a MEK inhibitor. Preferably, one or more MAPK inhibitors include BRAF inhibitors, particularly inhibitors of BRAF V600 variants. Preferably MAPK inhibitors include inhibitors of BRAF V600 variants, and / or The MAPK inhibitor in question includes a BRAF V600 variant inhibitor and / or a MEK inhibitor. Therefore, the present invention is preferably, Preferably, a cystathionine-γ-lyase (CSE) inhibitor for use in patients treated with a BRAF inhibitor to prevent or delay the onset or development of resistance to cancer treatment with a BRAF V600 inhibitor; and / or A cystathionine-γ-lyase (CSE) inhibitor for use in the treatment of a patient treated with a BRAF V600 inhibitor, wherein the patient is resistant to or at risk of developing resistance to one or more BRAF V600 inhibitors and MEK inhibitors; and / or A combination of a cystathionine-γ-lyase (CSE) inhibitor and a BRAF inhibitor for use in the treatment of cancer in patients, to prevent or delay the onset or development of resistance to the BRAF inhibitor. Regarding. Preferably, the cancer is a tumor, preferably a BRAF mutation-positive tumor, more preferably a BRAF V600 mutation-positive tumor. BRAF inhibitor resistance should preferably be understood as resistance to at least one of the one or more BRAF inhibitors, and includes resistance to each of the one or more BRAF inhibitors. Preferably, the patient is a mammalian patient, preferably a human patient, and preferably a patient with a BRAF V600 mutation-positive tumor.

[0030] 4. In preferred embodiments, the MAPK inhibitor, preferably the BRAF inhibitor, is an inhibitor of a BRAF V600 variant, the tumor is a BRAF V600 mutation-positive cancer, the mutation is preferably V600D, V600K, V600R, or V600E, more preferably V600K or V600E; more preferably V600E, Preferably, the patient is a mammalian patient, preferably a human patient. Preferably, the patient is resistant to or at risk of developing resistance to one or more BRAF V600 inhibitors, preferably inhibitors of BRAF V600 variants.

[0031] 5. In a preferred embodiment, the CSE inhibitor is for use in the patient to prevent or delay the onset and / or development of resistance to the cancer, preferably a tumor, and the tumor is a BRAF V600 mutation-positive cancer. The resistance is preferably acquired resistance. Preferably, the cancer or tumor is selected from the group defined in paragraph 2, and particularly preferably the cancer is melanoma. Preferably, the CSE inhibitor for use is defined in any of paragraphs 1-4 or as given herein. Preferably, the CSE inhibitor is selective for CSE or more selective for CSE than for CBS. Preferably, the tumor is a BRAF V600 mutation-positive cancer having a BRAF V600D, V600K, V600R, or V600E mutation. Preferably, the tumor or cancer is a melanoma.

[0032] 6. In preferred embodiments, the BRAF V600 variant inhibitors include vemurafenib (PLX4032), dabrafenib (GSK2118436), dabrafenib mesylate, encorafenib (LGX818), agerafenib (RXDX-105, CEP-32496), regorafenib (BAY73-4506), GDC-0879, RO5212054 (PLX3603), PLX-4720, PLX8394, and SB59088. 5. Select from L-779450, RAF265, RAF709, navolafenib (LXH254), LY3009120 (DP-4978), bevalafenib (HM95573), CH5126766 (RO5126766), TAK-632, rifirafenib (BGB-283), AZ628, AZ304, ARQ-736, XL281 (BMS-908662), or CCT196969. Inhibitors are understood to include any pharmaceutically acceptable salt, hydrate, or tautomer form, if present. Preferably, the BRAF inhibitor is a BRAF V600 inhibitor selected from vemurafenib (PLX4032), dabrafenib (GSK2118436), dabrafenib mesylate, encorafenib (LGX818), agerafenib (RXDX-105, CEP-32496), PLX-4720, PLX8394, RAF265, RAF709, LY3009120 (DP-4978), rifirafenib (BGB-283), AZ628, or AZ304. Preferably, the BRAF inhibitor is selected from vemurafenib, dabrafenib, or encorafenib. Preferably, the BRAF inhibitor is selected from vemurafenib, dabrafenib, or encorafenib, more preferably from vemurafenib or dabrafenib. Preferably, the present invention relates to a CSE inhibitor for use as described in any of paragraphs 3 to 5, wherein the BRAF inhibitor is selected from vemurafenib, dabrafenib, or encorafenib.

[0033] 7. In preferred embodiments, the MEK inhibitor is trametinib (GSK1120212), cobimetinib (XL518, GDC-0973), binimetinib (MEK162), selumetinib (AZD6244), mildametinib (PD-0325901), CI-1040 (PD184352), TAK-733, pimacertib (AS703026), refametinib, AZD8330, E6201, GDC-062 3. Select from RO-4987655 (CH4987655), CH5126766 (RO5126766), HL-085, SHR7390, TQ-B3234, CS-3006, FCN-159, WX-554, PD318088, PD98059, BI-847325, U0126, Myricetin, CInQ-03, G-573, PD184161, PD98059, RO5068760, or SL327. Preferably, the MEK inhibitor is selected from trametinib (GSK1120212), cobimetinib (XL518, GDC-0973), binimetinib (MEK162), or selumetinib (AZD6244), more preferably selected from trametinib, cobimetinib, or binimetinib, and more preferably the MEK inhibitor is trametinib. Preferably, the present invention relates to a CSE inhibitor for use according to any of paragraphs 3 to 5, wherein the MEK inhibitor is selected from trametinib, cobimetinib, binimetinib, or selumetinib, more preferably selected from trametinib, cobimetinib, or binimetinib, and more preferably the MEK inhibitor is trametinib.

[0034] 8. In one embodiment, particularly in embodiments according to any of paragraphs 3 to 7, the MAPK inhibitor includes BRAF inhibitors, particularly BRAF V600 variant inhibitors and MEK inhibitors, or combinations thereof. Preferably, the CSE inhibitor is used to prevent or delay the onset of acquired resistance to MAPK inhibitors in BRAF V600 mutant cancer, and preferably the MAPK inhibitor includes BRAF V600 inhibitors (particularly vemurafenib, dabrafenib, and encorafenib) and MEK inhibitors (particularly cobimetinib, trametinib, and binimetinib). Preferably, the combination of a BRAF inhibitor and a MEK inhibitor is selected from the group consisting of dabrafenib + trametinib, vemurafenib + cobimetinib, or encorafenib + binimetinib, more preferably dabrafenib + trametinib. Preferably, the BRAF inhibitor is selected from any of the groups listed in paragraph 6 above, and / or the MEK inhibitor is selected from any of the groups listed in paragraph 7 above. More preferably, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, or encorafenib, and the MEK inhibitor is selected from trametinib, cobimetinib, binimetinib, or selumetinib. Preferably, the combination of a BRAF inhibitor and a MEK inhibitor is dabrafenib + trametinib, vemurafenib + cobimetinib, or encorafenib + binimetinib, more preferably dabrafenib + trametinib.

[0035] 9. In preferred embodiments, particularly in embodiments according to any of paragraphs 3 to 8, the CSE inhibitor is selected from propargylglycine (PAG), β-cyanoalanine (BCA), L-aminoethoxyvinylglycine (AVG), hydroxylamine, I194496, I157172, S-3-carboxypropyl-L-cysteine ​​(CPC), NSC4056 (orintricarboxylic acid), L-aminoethoxyvinylglycine, 2-allylidene-hydrazinecarboditioate, or cystathionine-γ-lyase-IN-1 (CAS number 2165706-30-7). Preferably, the CSE inhibitor is intended for use in preventing or delaying the onset of acquired resistance to BRAF V600 and MEK inhibitors in BRAF V600 mutant cancers, and the inhibitor is selective for CSE. Preferably, the CSE inhibitor is selected from the group consisting of β-cyanoalanine (BCA), L-aminoethoxyvinylglycine (AVG), and propargylglycine (PAG), and more preferably from the group consisting of D,L-propargylglycine (2-aminopenta-4-ic acid or H-DL-Pra-OH) or N-propargylglycine (2-propyne-1-ylamino)acetic acid). Combined inhibition of BRAF and MEK results in a better therapeutic response, increased overall survival and progression-free survival, and reduced adverse effects by avoiding paradoxical activation of the MAPK pathway.

[0036] 10. In certain embodiments, the patient is a mammal, preferably a human.

[0037] 10a. In a preferred embodiment, the present invention relates to a CSE inhibitor for use in any of paragraphs 1 to 9, wherein the treatment prevents or delays the onset / development of resistance to BRAF inhibitor therapy, MEK inhibitor therapy, or combination therapy of a BRAF inhibitor and a MEK inhibitor in patients with BRAF V600 variants, preferably delaying the onset of resistance by at least 1, 2, 3, 4, 5, 6 months or more. In preferred embodiments, treatment comprising a CSE inhibitor and a MAPK inhibitor delays the onset of acquired resistance. Preferably, treatment comprising a CSE inhibitor and a MAPK inhibitor prevents the onset of acquired resistance. Preferably, treatment comprising a CSE inhibitor and a MAPK inhibitor increases the effectiveness of MAPK-targeted therapy.

[0038] 10b. In a preferred embodiment, the present invention provides a CSE inhibitor in combination with a MAPK inhibitor. Having a tumor, Patients who have not been treated previously, or Persons who have never been previously administered a MAPK inhibitor (BRAF inhibitor and / or MEK inhibitor), or Patients who have been previously treated with MAPK inhibitors (BRAF inhibitors and / or MEK inhibitors) but remain susceptible to MAPK inhibitors (BRAF inhibitors and / or MEK inhibitors), This relates to CSE inhibitors for use as prescribed in any of paragraphs 1-10, administered to [the patient]. Preferably, the tumor is one in which the serine-threonine protein kinase B-RAF (BRAF) contains the V600 mutation. More preferably, the mutations are V600, more preferably V600D, V600K, V600R, or V600E; more preferably V600K or V600E; more preferably V600E. In a preferred embodiment, the patient's tumor is selected from the group consisting of melanoma, colorectal cancer, colon cancer, rectal cancer, lung cancer, thyroid cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, gastrointestinal neuroendocrine tumor, neuroblastoma, glioma, astrocytoma, leukemia, hairy cell leukemia, hepatobiliary cancer, nephroblastoma (Wilms' tumor), histiocytosis, Langerhans cell histiocytosis, and Erdheim-Chester disease. Preferably, the tumor is melanoma, and more preferably BRAF V600 mutant melanoma (unresectable or metastatic melanoma, etc.).

[0039] 11. In preferred embodiments, the present invention relates to a CSE inhibitor for use in accordance with any of paragraphs 1 to 10 (including 10a or 10b), Here, CSE inhibitors are administered before the administration of MAPK inhibitors, particularly BRAF V600 variant inhibitors, or CSE inhibitors are administered concurrently with MAPK inhibitors, particularly BRAF V600 variant inhibitors, or CSE inhibitors are administered after the administration of MAPK inhibitors, particularly BRAF V600 variant inhibitors. Preferably, MAPK inhibitors, preferably BRAF V600 variant inhibitors and / or MEK inhibitors, more preferably combinations thereof, are administered separately, sequentially, simultaneously, or together with the CSE inhibitor.

[0040] 12. In preferred embodiments, the present invention relates to a CSE inhibitor for use according to any of paragraphs 1 to 11, wherein the CSE inhibitor and MAPK inhibitor are administered in sequential, intermittent, or continuous therapy. Preferably, the treatment comprises preventing or delaying the onset of acquired resistance to the MAPK inhibitor, wherein the CSE inhibitor and MAPK inhibitor are administered in sequential, intermittent, or continuous therapy. Preferably, the treatment is an intermittent therapy in which CSE inhibitors and MAPK inhibitors are administered for 5 weeks, followed by a 3-week period without administration, for example, a treatment period of 8 weeks. Preferably, the treatment is a series of therapies, and the CSE inhibitor and the MAPK inhibitor may be administered simultaneously / together for a certain period of time, or the CSE inhibitor and the MAPK inhibitor may be administered alternately. Preferably, the treatment is a series of therapies, with MAPK inhibitors (BRAF inhibitors and / or MEK inhibitors) administered to the patient daily and CSE inhibitors administered to the patient at least once a week, preferably at least twice a week.

[0041] 13. The present invention also relates to a combination, particularly a pharmaceutical kit, for use in the treatment of a patient's BRAF V600 mutation-positive cancer, preferably a BRAF V600 mutation-positive tumor, comprising a cystathionine-γ-lyase (CSE) inhibitor and a MAPK inhibitor in combination with (one or more) MAPK inhibitors, preferably for use in preventing or delaying the onset / development of resistance in the patient to treatment of said cancer with MAPK inhibitors. Preferably, the combination or kit comprises a CSE inhibitor, a BRAF inhibitor, particularly a BRAF V600 variant inhibitor and / or a MEK inhibitor, preferably a CSE inhibitor, a BRAF inhibitor and a MEK inhibitor, each as defined herein, for example in the paragraphs above. Preferably, the combination or kit is intended for use in a therapeutic method defined in paragraph 2 or paragraphs 3, 4, or 5 and / or paragraphs 10, 11, or 12. In a preferred embodiment, the combination or kit includes a cystathionine-γ-lyase (CSE) inhibitor in combination with a BRAF inhibitor as defined in paragraph 6 and / or a MEK inhibitor as defined in paragraph 7 and / or a combination as defined in paragraph 8. In any of these combinations in the preferred embodiment, the CSE inhibitor is as defined in paragraph 9.

[0042] 14. The present invention also relates to a pharmaceutical composition comprising a cystathionine-γ-lyase (CSE) inhibitor and a MAPK inhibitor for use in BRAF V600 mutation-positive cancer, particularly BRAF V600 mutation-positive cancer, and a pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition is for use in the treatment of a patient's cancer, preferably a tumor. Preferably, the pharmaceutical composition is for use in preventing or delaying the onset or development of resistance in the patient to treatment of the cancer with a MAPK inhibitor. Preferably, the pharmaceutical composition comprises a CSE inhibitor, a BRAF inhibitor, particularly a BRAF V600 variant inhibitor, and / or a MEK inhibitor, preferably a CSE inhibitor, a BRAF inhibitor, and a MEK inhibitor, as defined herein, for example, in the above paragraph. Preferably, the pharmaceutical composition is intended for use in a therapeutic method defined in paragraph 2 or paragraphs 3, 4, or 5 and / or paragraphs 10, 11, or 12. In a preferred embodiment, the combination or kit includes a cystathionine-γ-lyase (CSE) inhibitor in combination with a BRAF inhibitor as defined in paragraph 6 and / or a MEK inhibitor as defined in paragraph 7 and / or a combination as defined in paragraph 8. In any of these combinations in the preferred embodiment, the CSE inhibitor is as defined in paragraph 9.

[0043] 15. In further embodiments, the present invention relates to a method for treating a target cancer, preferably a BRAF V600 mutation-positive target tumor, preferably a patient's cancer, comprising administering to the target a cystathionine-γ-lyase (CSE) inhibitor in combination with a MAPK inhibitor. Preferably, the MAPK inhibitor is a BRAF inhibitor as defined herein, particularly a BRAF V600 variant inhibitor, or a MEK inhibitor, or a combination of a BRAF inhibitor and a MEK inhibitor. Preferably, the method is for preventing or delaying the onset or development of resistance in the patient to treatment of the cancer with the MAPK inhibitor. Preferably, the target is a patient diagnosed with a BRAF V600 mutation-positive cancer, preferably a tumor. In alternative embodiments, the present invention relates to a method for preventing or delaying the onset or development of resistance in a subject, preferably a patient, to the treatment of a tumor, the method comprising administering a cystathionine-γ-lyase (CSE) inhibitor to the subject in combination with a MAPK inhibitor. Preferably, the MAPK inhibitor is a BRAF inhibitor or MEK inhibitor as defined herein, or a combination of a BRAF inhibitor and a MEK inhibitor. Preferably, the subject is a patient diagnosed with BRAV V600 mutation-positive cancer, preferably a tumor. In the method of the present invention, cancer, preferably tumor, contains the V600 mutation (BRAF V600 mutation) in the serine-threonine protein kinase B-RAF. More preferably, the mutations are V600D, V600K, V600R, or V600E; even more preferably V600K or V600E; and very preferably V600E. In a preferred embodiment, the patient's tumor is selected from the group consisting of BRAF V600 variant melanoma, colorectal cancer, colon cancer, rectal cancer, lung cancer, thyroid cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, gastrointestinal neuroendocrine tumor, neuroblastoma, glioma, astrocytoma, leukemia, hairy cell leukemia, hepatobiliary tract cancer, nephroblastoma (Wilms' tumor), histiocytosis, Langerhans cell histiocytosis, and Erdheim-Chester disease. Preferably, the tumor is melanoma, and more preferably BRAF V600 mutant melanoma (unresectable or metastatic melanoma, etc.). Preferably, the combination or kit defined in paragraph 13 or the pharmaceutical composition defined in paragraph 14 is administered to the subject by a therapeutic method defined in paragraph 2 or paragraphs 3, 4, or 5 and / or paragraphs 10, 11, or 12. Preferably, the subject is a mammal, preferably a human patient, as preferably defined in paragraph 10. In a preferred embodiment, the method includes detecting the presence of BRAF mutations in a tumor sample derived from the subject prior to the administration step. Therefore, preferably, a diagnostic step is performed to detect the presence or absence of BRAF mutations in the tumor sample before initiating combination therapy. Preferably, BRAF mutations (preferably V600 mutations) are determined using a method comprising (a) performing PCR or sequencing on nucleic acids (e.g., DNA) extracted from a sample of the patient's melanoma, and / or (b) determining the expression of BRAF mutant proteins in the sample, and / or using Sanger sequencing. Preferably, the BRAF mutation (preferably the V600 mutation) is as defined in paragraph 10. In a preferred embodiment, the patient is sensitive to MAPK inhibitor therapy, preferably BRAF inhibitors and / or MEK inhibitors, or a combination thereof.

[0044] 16. In a preferred embodiment of the treatment method, CSE inhibitors are administered before the administration of MAPK inhibitors, particularly V600 variant inhibitors, or CSE inhibitors are administered concurrently with MAPK inhibitors, particularly V600 variant inhibitors, or CSE inhibitors are administered after the administration of MAPK inhibitors, particularly V600 variant inhibitors. Preferably, MAPK inhibitors, preferably BRAF inhibitors, particularly BRAF V600 variant inhibitors, and / or MEK inhibitors, preferably in combination thereof, are administered separately, sequentially, simultaneously, or together with the CSE inhibitor. Preferably, BRAF inhibitors, particularly BRAF V600 variant inhibitors, and MEK inhibitors are administered simultaneously, preferably from the start of treatment, and especially as soon as possible after diagnosis. Preferably, treatment with a CSE inhibitor is initiated within one month, preferably within one week, preferably within 6, 5, 4, 3, 2, or 1 day after the patient is diagnosed with BRAF600 mutation-positive cancer. Preferably, the CSE inhibitor in combination with a MAPK inhibitor is administered to patients with tumors that have not been previously treated, have not been previously administered a MAPK inhibitor (BRAF inhibitor and / or MEK inhibitor), or have been previously treated with a MAPK inhibitor (BRAF inhibitor and / or MEK inhibitor) but remain sensitive to the MAPK inhibitor (BRAF inhibitor and / or MEK inhibitor). In preferred embodiments, cancer, preferably tumor, is melanoma, skin cancer, epithelial cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, thyroid cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, gastrointestinal neuroendocrine tumor, neuroblastoma, glioma, astrocytoma, leukemia, hairy cell leukemia, hepatobiliary cancer, nephroblastoma (Wilms' tumor), histiocytosis, Langerhans cell histiocytosis and Erdheim-Chester disease. Preferably melanoma, colorectal cancer, colon cancer, rectal cancer, lung cancer, CSE inhibitors, selected particularly from the group consisting of melanoma, for use according to paragraph 1.

[0045] 17. In preferred embodiments, the present invention relates to a CSE inhibitor for use according to any of paragraphs 1 to 10, wherein the CSE inhibitor and MAPK inhibitor are administered in sequential, intermittent, or continuous therapy. Preferably, the treatment is an intermittent therapy in which CSE inhibitors and MAPK inhibitors are administered preferably daily for 5 weeks, followed by a 3-week period without administration, for example, an 8-week treatment period. Preferably, the treatment is a series of therapies, and the CSE inhibitor and the MAPK inhibitor may be administered simultaneously / together for a certain period of time, or the CSE inhibitor and the MAPK inhibitor may be administered alternately. In preferred embodiments, the treatment prevents or delays the onset / development of resistance to BRAF inhibitor therapy, MEK inhibitor therapy, or combination therapy of BRAF inhibitors and MEK inhibitors, preferably delaying the development of resistance by at least one, two, or three months. In preferred embodiments, treatment comprising a CSE inhibitor and a MAPK inhibitor delays the onset of acquired resistance compared to MAPK therapy alone. Preferably, treatment comprising a CSE inhibitor and a MAPK inhibitor prevents the onset of acquired resistance. Preferably, treatment comprising a CSE inhibitor and a MAPK inhibitor increases the effectiveness of MAPK-targeted therapy.

[0046] definition As used herein, “subject” refers to an individual of an animal species, preferably a vertebrate, more preferably a mammalian species, and very preferably the individual is a primate, hominin, or human. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; domestic animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. In one embodiment, the mammal is human.

[0047] "Patient" refers to an object that is currently receiving, or intends to receive, medical or veterinary observation, supervision, diagnosis, or treatment.

[0048] A "neoplasm" is a type of abnormal, excessive, or uncontrolled growth of tissue within a body. (The process that leads to the formation or production of a neoplasm is called "neoplasmogenesis.") Neoplasmic growth does not coordinate with the normal growth of surrounding tissue and continues to grow abnormally even after the original trigger is removed. Numerous types of neoplasms are listed in (Kumar et al., 2017).

[0049] A "tumor" is a neoplasm that forms a mass of cells with abnormal growth. In one embodiment, the tumor is formed by a malignant neoplasm and is therefore a malignant tumor. Preferably, the tumor is a solid tumor.

[0050] The term "cancer" is understood herein to mean a malignant neoplasm caused by the uncontrolled division of neoplastic cells in a part of the body, preferably one that is ready to spread to other parts of the body, i.e., to form cancer in different tissues (metastasis). Preferably, cancer is a tumor.

[0051] The term "melanoma" refers to a type of cancer that arises from pigment-producing cells known as melanocytes. In certain embodiments, melanoma is a type of skin cancer.

[0052] The term “inhibitor” as used herein refers to a substance, particularly a molecular substance or molecule, that reduces or diminishes, for example, the activity of a protein in the body of interest, or blocks it. Such a protein is the target of the inhibitor. In a preferred embodiment, the inhibitor is an inhibitor of a signaling pathway, a substance that reduces or diminishes, for example, the signal transmitted from one molecule to another within a cell. In a particular embodiment, the signaling inhibitor is involved in the process of cancer. The terms “inhibit,” “inhibit,” or “inhibit” refer to an effect, preferably the effect of the inhibitor on a target protein.

[0053] The terms "BRAF V600" or "V600" refer to a mutation in the BRAF gene in which the valine (V) at position 600 is replaced by another amino acid. For example, "BRAF V600E" or "V600E" is a mutation in which the valine (V) at position 600 is replaced by glutamic acid (E). V600E is a driver mutation in certain diseases such as melanoma, hairy cell leukemia, colorectal cancer, and non-small cell lung cancer.

[0054] A "BRAF inhibitor" is a chemical substance, drug, or compound that inhibits serine / threonine protein kinase B-Raf and / or its variants. BRAF inhibitors can preferably inhibit the V600 variant B-Raf.

[0055] A "MEK inhibitor" is a chemical substance, drug, or compound that inhibits the mitogen-activated protein kinase kinase enzymes MEK1 and / or MEK2. MEK inhibitors can be used to affect the MAPK / ERK pathway.

[0056] A "CSE inhibitor" is a compound, chemical, or drug that inhibits the cystathionine-γ-lyase (CSE) protein. The term "CSE inhibitor" encompasses complete or partial inhibitors of CSE enzyme activity in the synthesis of hydrogen sulfide.

[0057] "Selective CSE inhibitor" is understood herein as a CSE inhibitor that is selective to CSE more than at least other enzymes involved in cancer development. For example, a CSE inhibitor that is selective to inhibit CSE against enzymes selected from the group consisting of PSAT1, VEGFR, PDGFR, c-Kit, and RET.

[0058] In very specific embodiments, the “selective CSE inhibitor” is selective for CBS.

[0059] The “composition” of the present invention is a composition of substances comprising at least one compound of the present invention as an activator and at least one further substance. Preferably, the compound of the present invention is present in an effective amount. The composition may also contain further bioactive substances useful, for example, in combination therapy. Furthermore, the composition may contain bioacceptable carriers, formulations, excipients, etc., that may be known in the art. Pharmaceutical compositions include any composition intended for the treatment of a target, and in particular include pharmaceuticals, drugs, tailored pharmaceuticals and compounded drugs.

[0060] The term "treatment" in this document refers to any process, action, and especially treatment in which the subject or patient receives support, in particular medical or veterinary support, either directly or indirectly, for the purpose of improving the condition of the subject or patient. Improving the condition of the subject may include restoring or maintaining the normal function of an organ or tissue, and may include partial improvement, improvement or reduction of a disease condition, in particular cancer, in particular melanoma. Treatment typically refers to the administration of an effective amount of the compounds or compositions described herein, in particular combinations of the compounds disclosed herein. Unless otherwise specified, therapeutic treatment includes both medical or veterinary treatment and prevention (or prevention), i.e., the onset or prevention of the onset of a condition, e.g., resistance to treatment or treatment.

[0061] The term "treatment" means the treatment of a disease, disorder, or condition.

[0062] The term "combination therapy" refers to a treatment that combines two or more treatment methods. For example, it is a treatment in which a patient is given two or more drugs or medications for a single disease or disorder.

[0063] The “combination” according to the present invention is understood to comprise at least two activators (preferably inhibitors in the present invention) for administration to a subject or patient. The activators of the combination may be administered simultaneously or sequentially, or in any therapeutic regimen useful for providing effective amounts from both or each of the therapeutic agents.

[0064] As used herein, "concurrent administration," etc., means the administration of selected therapeutic agents to a single patient and is intended to include therapeutic regimens in which the drugs are administered by the same or different routes of administration, or at the same or different times.

[0065] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount of an administered drug or compound that reduces, to some extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system, particularly cancer or tumor. For example, an “effective dose” for therapeutic use is the amount of a composition containing the compounds disclosed herein that is necessary to provide a clinically significant reduction in disease symptoms, preferably regression of cancer or tumor, particularly melanoma. The appropriate “effective” dose in an individual case may be determined using techniques such as dose escalation studies.

[0066] The term "effective dose" refers to the amount of compound in a composition that is necessary to exert the effect of the activator. "Therapeutic effective dose" is sufficient to alleviate or prevent (or prevent the worsening of) one or more symptoms or characteristic parameters of a condition, such as a disorder or disease.

[0067] The singular forms "a," "an," and "the," or at least "a" and "an," imply multiple references unless explicitly indicated otherwise in the context.

[0068] The terms “comprises,” “comprising,” or “including” should be interpreted here as having a non-exclusive meaning, allowing for the addition or involvement of further features, methods, processes, or components to those that include the listed features, methods, processes, or components. “Comprising” can be replaced with “including” if the implementation of a given linguistic variation requires it, and can be limited to “consisting essentially of” if other components or components are not essential for carrying out the invention.

[0069] Abbreviation A375-X A375 cell line derived xenograft ABCG2 ATP-binding cassette (ABC) transporter G family member 2 Akt Protein Kinase B APOE (Apolipoprotein E) Asn asparagine Asp Aspartic acid ATP6V0D2 V-type proton ATPase subunit d 2 Braf Serine / Threonine Protein Kinase B-raf CARS2 cysteine-tRNA ligase, mitochondria CBS Cystathionine-β-Synthase CDO (Cysteine ​​Dioxygenase) CYP Cytochrome P450 Cys cysteine Cys-SSH cysteine ​​persulfide CySSyC Cystine CSAD Cysteine ​​Sulfinate Decarboxylase CSE (Cystathionine-γ-lyase) CTH (Cystathionine) The dihydrolipoyllysine residue succinyltransferase component of the DLST 2-oxoglutarate dehydrogenase complex. DT dabrafenib-trametinib DTR dabrafenib-trametinib resistance ECAR extracellular acidification rate ERK Mitogen-Activated Protein Kinase Kinase ETHE1 persulfide dioxygenase G6PD Glucose-6-phosphate dehydrogenase GAPDH Glyceraldehyde-3-phosphate dehydrogenase GCLC Glutamate-Cysteine ​​Ligase Catalyst Subunit gGluCys (gamma-glutamyl-cysteine) Gln (Glutamine) GLS Glutaminase Glu Glutamate GLUL (Glutamine Synthetase) GOT aspartate aminotransferase GPX Glutathione Peroxidase GS Glutathione Synthase GSH Glutathione GSSG Oxidized Glutathione GSSH Glutathione Hypersulfide H2S Hydrogen sulfide H2S2 Hydrogen disulfide HCys Homocysteine Hlanth homolanthionine HMW High molecular weight HSPB1 Heat shock protein B1 IDH (isocitrate dehydrogenase) Lanth Lunchion LMW low molecular weight MAPK Mitogen-Activated Protein Kinase MEK Mitogen-Activated Protein Kinase Kinase Met Methionine MPST (Mercatopyruvate Sulfurtransferase) Nrf2 (Nuclear Factor Red Blood Cell II-Related Factor II) OCR oxygen consumption rate OGDH 2-oxoglutarate dehydrogenase complex OXPHOS (Oxidative Phosphorylation) PAG D,L-propargylglycine PDH pyruvate dehydrogenase PDX patient-derived xenografts PFKFB 6-phosphofructose-2-kinase / fructose-2,6-biphosphatase Pyr Pyruvate Ras Ras GTP Arze ROS (Reactive Oxygen Species) SDHA (Succinate Dehydrogenase Flavoprotein Subunit A) Serine SO sulfite oxidase SOD Superoxide Dismutase SQOR Sulfide:Quinone Oxidation Reductase TRP14 Thioredoxin Domain-Containing Protein 17 Trx Thioredoxin TrxR1 Thioredoxin Reductase 1 TST thiosulfate sulfurtransferase xCT cystine / glutamate transporter [Brief explanation of the drawing]

[0070] [Figure 1]This figure shows that dabrafenib-trametinib-resistant A375 cells overexpress genes involved in drug resistance and genes involved in redox balance. In addition to activating the PI3K / Akt pathway, dabrafenib-trametinib-resistant cells overexpress ABC transporters and cytochrome P450 proteins. (A) To investigate the molecular and metabolic background of melanoma resistant to MAPK inhibitors (MAPKi), we created a DT-resistant line of BRAF V600E mutant A375 human melanoma cells (A375-DTR) by long-term culture in the presence of escalating doses of dabrafenib (BRAF inhibitor) and trametinib (MEK1 / 2 inhibitor). Figure A is a simplified diagram showing how dual inhibition of V600E mutant Braf by dabrafenib and MEK1 / 2 by trametinib leads to blockade of the MAPK-ERK pathway. (B) The viability of resistant cells in the presence of the drug was tracked using a proliferation assay. A375 control and DTR cells were cultured in or without DT. Plates were fixed daily, and relative cell mass was measured by a sulforhodamine B (SRB) assay. Dilution and path length corrected absorbance values ​​are shown. (C) The activity of the MAPK-Erk pathway was investigated by measuring the amounts of phospho-MEK1 / 2 and phospho-ERK1 / 2 in A375 control cells and DT-resistant (DTR) cells cultured for 0, 24, 48, or 72 hours in or without DT by Western blot analysis. β-actin was used as a loading control. (D) The level of phosphorylated Akt was measured by Western blot analysis in A375 control cells and DT-resistant (DTR) cells cultured for 0, 24, 48, or 72 hours in or without DT. Inhibition of the MAPK-ERK pathway resulted in increased Akt phosphorylation (Figure 3A), which was consistent with previous reports demonstrating that increased activity of the PI3K-Akt pathway contributes to DT resistance (Liu et al., 2020; Sun et al., 2014). β-actin was used as a loading control. Using a real-time quantitative PCR (RT-qPCR) based array of (E-F)Qiagen, we identified overexpression of several genes involved in the development of cancer drug resistance.Gene expression in untreated control A375 cells and DT-treated DTR A375 cells was compared using Qiagen RT-qPCR-based arrays named Human Drug Metabolism (E) and Cancer Drug Resistance (F). (G) Upregulation of genes involved in xenobiotic neutralization was measured by RT-qPCR using custom-designed oligonucleotides. Values ​​were normalized for GAPDH, β-actin, and b2M. Multiple increases compared to untreated controls were calculated. (H-J) Levels of proteins involved in redox balance were measured by Western blot analysis in A375 control cells and DT-resistant (DTR) cells cultured in the presence or absence of DT. GAPDH was used as a loading control. Each Western blot experiment was repeated at least three times, and representative images are shown. *p<0.05, **p<0.01, ***p<0.001 (by standard one-way ANOVA or Brown-Forsyth test) (n=2 technical replicates from 3 biological replicates (E)). [Figure 2]Altered energy metabolism in DT-treated cells and DT-resistant (DTR) cells. (A) Mitochondrial function of cells was analyzed by measuring the rate of oxygen consumption (OCR) of cells using a Seahorse Cell Analyzer with the Mito Stress Test kit. After the assay, cells were fixed and total protein levels were measured by sulfolodamine B (SRB) assay. Measured OCR values ​​were normalized to dilution and path length corrected absorbance. (B) Glycolytic activity of cells was analyzed by measuring the rate of extracellular acidification (ECAR) using a Seahorse Cell Analyzer with the Glycolysis Stress Test kit. After the assay, cells were fixed and total protein levels were measured by sulfolodamine B (SRB) assay. Measured ECAR values ​​were normalized to dilution and path length corrected absorbance. (C) Protein levels of citrate cycle enzymes involved in NADH production were measured by Western blot analysis in A375 control cells and DT-resistant (DTR) cells cultured in or without DT. DT-treated control cells were treated with DT for 6 days. GAPDH was used as a loading control. (D-E) Amino acid levels from cell lysates (D) and cell culture medium (E) were measured using the EZ:fast amino acid analysis kit after 48 hours of culture in or without DT, followed by LC-MS / MS analysis. Amino acid levels in lysates were normalized to total protein content, and medium measurements were compared to fresh medium. (F) Simplified diagram showing metabolic pathways involved in glutamate (Glu) conversion. (G) Gene expression was analyzed by RT-qPCR. Detected values ​​were normalized to GAPDH, β-actin, and b2M. The multiplier increase compared to the untreated control was calculated. (H) Protein levels were analyzed by Western blotting in A375 control cells and DT-resistant cells cultured in or without DT. DT-treated control cells were treated with DT for 6 days. Protein levels of GOT1 and GOT2 were measured by Western blotting. GAPDH was used as a loading control. Each Western blotting experiment was repeated at least three times, and representative images are shown.*p<0.05, **p<0.01, ***p<0.001, representative figures from n=15 technical replicates from two independent experiments (A, B) and n=2 technical replicates from three biological replicates (D, E, G) by standard one-way ANOVA or Brown-Forsyth test (A, B, D, E, G). [Figure 3]Dabrafenib-trametinib-resistant A375 cells, which overexpress genes involved in the redox balance of cysteine ​​metabolism, are reprogrammed in DT-resistant melanoma cells due to DT treatment. (A) Cells were cultured with different concentrations of cystine, with or without DT and with or without 100 nM sodium selenite. Cell viability was measured after 72 hours using an SRB assay. Dilution and path length corrected absorbance values ​​are shown. (B) Cells were cultured for 24, 48, or 72 hours in the absence of sodium selenite (Se), lysed, and selenium protein levels were measured by Western blot analysis. (C) Cystine (CySSyC) and glutamic acid (Glu) levels were measured from the culture medium on the cells after 72 hours of culture using the EZ:fast amino acid analysis kit. Changes in CySSyC and Glu levels were compared to fresh culture medium. Multiple increases compared to untreated controls were calculated. A negative value indicates a decreased level (uptake) compared to fresh medium, and a positive value indicates an increased level (outflow). (D) Glutathione levels were measured from cell lysates using HPE-IAM alkylating agent followed by LC-MS / MS detection. The measured values ​​were normalized to total protein content, and the multiplier compared to the untreated control was calculated. (E) GSTpi levels were measured by Western blot analysis in A375 control cells and DT-resistant (DTR) cells cultured in or without DT. GAPDH was used as a loading control. (F) Simplified diagram showing the source and sink of intracellular cysteine ​​(Cys). (G) LMW cysteine, cystine, and cysteine ​​persulfide levels were measured from cell lysates using HPE-IAM alkylating agent and LC-MS / MS detection. The measured values ​​were normalized to total protein content, and the multiplier compared to the untreated control was calculated. (H) Gene expression was analyzed by RT-qPCR. Data were normalized for GAPDH, β-actin, and b2M. The multiplier of increase compared to untreated controls was calculated.(I) Levels of proteins involved in Cys metabolism were measured by Western blotting in A375 control cells cultured in or without DT, as well as in DT-resistant (DTR) (I / 1) and SK-MEL28 (I / 2) cells. (J) Levels of LMW thiols and hypersulfides were measured by alkylation with HPE-IAM followed by LC-MS / MS detection. Detected values ​​were normalized to total protein levels, and the multiplier compared to untreated controls was calculated. Each Western blot was repeated at least three times, and representative images are shown. GAPDH was used as a loading control. *p<0.05, **p<0.01, ***p<0.001, by standard one-way ANOVA or Brown-Forsyth test (A, C, D), 4 biological replicates to n=2 replicates (A), 2 biological replicates to n=2 replicates (C), and 3 biological replicates to n=3 replicates (D); and *p<0.05, **p<0.01, ***p<0.001, by standard one-way ANOVA or Brown-Forsyth test (G, H, J), 3 biological replicates to n=3 technical replicates (G, J), and 3 biological replicates to n=2 technical replicates (H). [Figure 4]Enzymatic function of CSE and CBS and analysis of enzymes in the sulfide catabolism pathway associated with A375 cells. (A) Total steady-state levels of cysteine ​​metabolic intermediates were measured by alkylation with HPE-IAM followed by LC-MS / MS detection. Detected values ​​were normalized to total protein content. Multiple increases compared to untreated A375 controls are shown. (B) Chemical reactions of CBS and CSE proteins were tracked using stable methionine isotopes. Heavy atoms are marked with asterisks. (C-D) Cells were treated with heavy Met(34S) for 0, 18, 24, and 48 hours, and lysed analytes, as well as normal and heavy analytes, were measured using the EZ:fast amino acid kit followed by LC-MS / MS detection. Detected values ​​were normalized to total protein content. Methionine-derived heavy analytes are marked with an asterisk (*). Ratios of heavy / total (C) and steady-state levels (D) are shown. (E) Simplified diagram showing the canonical and sulfide / persulfide formation reactions of CSE and CBS. (F) Chemical reactions of CBS and CSE proteins were tracked using stable cystine isotopes. Heavy atoms are marked with stars. (G~H) Cells were treated with heavy CySSyC (13C, 15N) for 0, 18, 24 or 48 hours, lysed, and measured using the EZ:fast amino acid kit, followed by LC-MS / MS detection. Detected values ​​were normalized to total protein content. Heavy analytes derived from CySSyC are signed with *. Heavy / total ratio (G) and steady-state levels of heavy analytes (H) are shown. (I) Persulfide formation of Cys residues of proteins as measured by alkylation with HPE-IAM and subsequent LC-MS / MS detection. A multiplier increase compared to untreated controls is shown. Standard one-way ANOVA or Brown-Forsyth tests from n=3 technical replicates from 3 biological replicates (A) and n=3 replicates from 2 biological replicates (I) yielded *p<0.05, **p<0.01, and ***p<0.001. [Figure 5]Cys and H2S metabolism are rewired in vemurafenib-treated and resistant melanoma cells. (A) Simplified diagram showing how the Braf V600E inhibitor vemurafenib blocks the MAPK / ERK pathway. (B) To determine whether our findings regarding cys and H2S metabolic rewiring are a common adaptive response in melanoma cells to Braf V600E inhibition, we created vemurafenib-resistant A375 cell lines by long-term culture of cells in the presence of escalating doses of vemurafenib. Vemurafenib (V)-treated A375 control and vemurafenib-resistant (VR) cells were grown in or without V. After 72 hours, the cells were fixed and the relative cell mass was measured by an SRB assay. (C) V-treated control cells were cultured with V for 6 days. By measuring MEK1 / 2 and ERK1 / 2 phosphorylation, the inventors found that V treatment effectively blocked MEK1 / 2 phosphorylation, and that MEK1 / 2 phosphorylation was completely restored in VR cells. Protein levels of enzymes involved in redox balance, Cys, and H2S metabolism were measured by Western blot analysis from untreated, V-treated, and V-resistant cells. GAPDH was used as a loading control. (D) Intracellular levels of LMW thiols were measured by alkylation with HPE-IAM followed by LC-MS / MS detection. Measurements were normalized to total protein content. Multiple increases or ratios compared to the untreated control are shown. Each Western blot was repeated at least three times, and representative images are shown. *n=3 from 3 biological replicates, and p<0.05, **p<0.01, ***p<0.001 (B, D) by standard one-way ANOVA or Brown-Forsyth test from 3 technical replicates. [Figure 6]Metabolic analysis (analysis of LMW thiols and persulfides) of DT-treated cell line-derived and patient-derived xenograft tumors. (A) Xenograft tumors derived from the A375 cell line (A375-X) were grown in NOD SCID mice and treated orally with DT for 4 days. Mice were sacrificed 24 hours after the last treatment and tumor weight was measured. (B) Snap-frozen homogenized A375 xenograft tumor samples were alkylated and dissolved with ice-cold HPE-IAM / methanol. LMW thiols were measured by LC-MS / MS detection. The multiplier increase compared to untreated controls was calculated. (C) Patient-derived xenograft (PDX) tumors were grown in NOD SCID mice and treated orally with DT for 6 days four times. Mice were sacrificed 24 hours after the last treatment and tumor weight was measured. (D) Snap-frozen homogenized patient-derived xenograft (PDX) tumor samples were alkylated with ice-cold HPE-IAM / methanol and dissolved. LMW thiols were measured by LC-MS / MS detection. Multiple increases compared to untreated controls were calculated. *p<0.05, **p<0.01, ***p<0.001 (by standard one-way ANOVA or Brown-Forsyth test from n=6 biological replicates). [Figure 7]Higher levels of hypersulfide are preferred for DT-treated A375 cells. Combinations of Braf and / or MEK inhibitors with CSE inhibitors yield a more effective therapeutic response. (A-B) Protein levels of sulfide catabolism pathway enzymes measured by Western blot analysis in control (cultured for 6 days with or without DT) and DTR cells (A) with or without DT, and control (cultured for 6 days with or without V) and VR cells. GAPDH was used as a loading control. (C) Thiosulfate (S2O3) levels per unit of total protein, measured using a monobromobiman-based alkylation protocol followed by fluorescence detection. (D) ETHE1 protein levels in sh control (marked with C) and shETHE1 single clones (1-9) were measured by Western blot. Cells grown from clone 9 were used for further experiments. (E) Ethe silenced (shETHE1) and lentiviral control (sh control) A375 cells were cultured with or without DT for 6 days and then lysed. Levels of proteins involved in Cys and H2S metabolism were analyzed by Western blotting. GAPDH was used as a loading control. (F) Proliferation of untreated (7F / 1) and DT-treated (7F / 2) sh controls and shETHE1 cells was measured by SRB assay. Dilution and path length corrected absorbance values ​​are shown. (G) Intracellular levels of LMW thiols and H2S metabolic intermediates were measured from cell lysates using HPE-IAM alkylating agent followed by LC-MS / MS detection. Measurements were normalized to total protein content and the multiplier compared to the untreated control was calculated. (H) Extracellular levels of GSSH were measured from cell culture medium and compared to fresh medium using HPE-IAM alkylating agent followed by LC-MS / MS detection. (I) Protein persulfideation was measured from cell lysates using HPE-IAM alkylating agent followed by LC-MS / MS detection. [Figure 8]This figure shows that CSE is upregulated in tumors from melanoma patients treated with DT. (A) CSE levels were measured by immunohistochemistry from malignant cutaneous melanomas removed from the same patient before (left) and after (right) DT treatment. (B) CSE levels measured by immunohistochemistry in lymphoid melanoma metastases from the same patient before (left) and after (right) DT treatment. (C) CSE levels in tumors removed from different patients before (unbounded) and after (bounded) DT treatment. [Figure 9] Treatment-naive melanoma cells are not sensitive to PAG and AOAA treatment. (A) Treatment-naive A375 cells were treated with 0.5 mM PAG for 9 days. Culture plates were fixed daily, and proliferation was measured by SRB assay. (B) Treatment-naive A375 cells were treated with 250 or 500 μM AOAA for 48 hours. Relative cell volume was measured by SRB assay. [Figure 10]PAG-mediated inhibition of CSE delays the onset of acquired resistance to MAPK inhibitors in vitro. (A-B) A375 cells were cultured for 2 months with vemurafenib (A) or dabrafenib-trametinib (B) in and without 0.5 mM PAG. Fresh medium was added every 3 days. The proliferation of V-treated cells and V+PAG-treated cells was compared to untreated A375 cells (A). The proliferation of DT-treated cells compared to DT+PAG-treated cells was compared to DT-resistant and untreated A375 cells (B) in a 4-day proliferation assay. Plates were fixed daily and SRB assays were performed. Dilution and path length corrected absorbance values ​​are shown. (C) A375 cells were cultured for 16 weeks with encorafenib (BRAF V600E inhibitor) and binimetinib (MEK1 / 2 inhibitor) (EB) in and without 0.5 mM PAG. The proliferation of EB-treated cells was compared to that of EB+PAG-treated cells using a 7-day proliferation assay. Plates were fixed daily, and SRB assays were performed. Dilution and path length corrected absorbance values ​​are shown. (D) A375 cells were cultured for 10 weeks with cobimetinib (MEK1 / 2 inhibitor) and vemurafenib (Braf V600E inhibitor) (CV) in and without 0.5 mM PAG. Representative microscopic images are shown. (E~H) Phospho-MEK1 / 2, phospho-ERK1 / 2, and phospho-Akt levels were measured in A375 cells cultured with or without PAG for 2 months, or with EB for 10 weeks (G) or 16 weeks (H), or with V(E) or DT(F), and compared with untreated controls (E~H) and previously prepared DT-resistant strains marked with R(F). Each Western blot was repeated at least three times, and representative images are shown. GAPDH was used as the loading control. [Figure 11]Inhibition of CSE delays the onset of acquired resistance to dabrafenib-trametinib (DT) therapy in vivo. (A) A xenograft model with the A375 melanoma cell line has been established. Tumor-bearing mice were randomized and treated with DT five times a week. Mice in the DT+PAG group received PAG treatment three times a week in addition to DT treatment. Tumors were measured two or three times a week by manual caliper. (B) Progression-free survival was calculated for each group. Treatment was considered ineffective if the tumor volume exceeded that of the pre-treatment volume. (C) If 11 out of 12 mice in the DT group were resistant, the mice were sacrificed and tumor weight was measured. [Figure 12] Preparation of CSE knockout cell lines using CRISPR-Cas9 technology. (A) CSE levels were measured from CRISPR control and CSE knockout A375 cell lines with and without 48-hour DT treatment. Knockout was successful, and CSE levels were barely detectable in the CSE knockout cell population. 48-hour DT treatment significantly increased CSE levels in control cells, but only low background levels were detected in CSE KO cells. (B) CSE levels were measured from CRISPR control and CSE knockout A375 cell lines 1.5 months after DT treatment. No significant difference was observed. GAPDH was used as a loading control. [Modes for carrying out the invention]

[0071] The inventors have demonstrated the positive effect of CSE inhibitors in counteracting cancer or tumor resistance to MAPK inhibitor therapy, particularly in the treatment of cancer with the Braf V600 mutation. The cancer is preferably melanoma.

[0072] Specific results from prior art [Sun et al: Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis, Hepatology, 64(2), 488-500 (2016)] suggest that PAGs (CSE inhibitors) may enhance the effects of kinase inhibitors such as sorafenib in certain cancers, such as hepatocellular carcinoma (HCC).

[0073] However, the inventors tested PAG and the nonspecific CSE inhibitor AOAA in BRAF V600 mutant melanoma, in which these compounds did not exhibit antiproliferative effects.

[0074] Surprisingly, the inventors have found that co-administration of the CSE inhibitor D,L-propargylglycine (PAG) with DT, V, CV, or EB effectively delays the onset of acquired drug resistance, thereby providing a more efficient combination therapy for patients with BrafV600 mutations, particularly the BrafV600E mutation, in melanoma patients and others.

[0075] The inventors carefully analyzed the altered energy landscape and redox environment in dabrafenib / trametinib treatment (DT treatment) or vemurafenib treatment (V treatment) controls, as well as the crucial roles and novel mechanistic details of reprogrammed cysteine ​​and hydrogen sulfide metabolism in cellular adaptation (both in vitro and in vivo) to these drugs in resistant (VR or DTR) melanoma cells, DT-treated cells, and DTR cells, respectively. These results were also confirmed in vivo by trans-sulfur metabolome analysis of treated, untreated, and patient-derived xenograft mouse models. Surprisingly, among many immediate adaptive changes to treatment, elevated CSE expression was found to play a critical role in cancer cell survival and the development of drug resistance during DT or V treatment. Elevated CSE expression resulted in increased GSH and protein cys persulfide formation, which supported cancer cell survival and tumor growth among other factors by providing additional protection against Braf mutations, particularly BrafV600E inhibition-induced oxidative stress. Increased RSS production is also utilized to re-regulate mitochondrial energy production in melanoma cells to compensate for the elevated energy requirements of cells in the presence of these drugs, enabling them to survive and proliferate. Our targeted metabolome analysis revealed several key details on how altered trans-sulfurization relates to the TCA cycle or other metabolic processes such as glutaminolysis, further supporting cytoprotection.

[0076] More precisely, the inventors found that the increase in oxidative stress upon inhibition of the MAPK / ERK pathway in melanoma cells by DT treatment is counteracted by Nrf2-mediated overexpression of several antioxidant genes. In DT-resistant cells, a more balanced redox environment and restoration of glycolysis are predicted based on the expression patterns of enzymes that regulate the cellular antioxidant response and glucose metabolism.

[0077] Further experiments revealed that metabolic pathways involved in cellular energy theory are reprogrammed in melanoma cells resistant to BrafV600Ei and MEK1 / 2i. Along with upregulation of OXPHOS accompanied by increased expression of the NADH-producing CAC enzyme (for fueling the electron transport chain), we found increased glutaminolysis in DT-treated and DT-resistant cells, which indicates an increased demand for Glu.

[0078] DT-treated cells and DT-resistant cells also require increased levels of CySSyC to counteract oxidative stress and promote GSH synthesis, which is used to neutralize anticancer drugs.

[0079] Returning to the inhibition of the MAPK / Erk pathway by DT, inhibition in control cells resulted in an immediate increase in CSE levels and a decrease in CBS levels. Surprisingly, however, the opposite pattern was observed in DT-resistant cells, with restored CBS expression and a reduction in CSE to near-undetectable levels below untreated control levels. The elevated H2S2 levels in DT-resistant cells, as well as the higher concentrations of H2S and GSSH in both DT-treated and DTR cells, suggested that the changes in CSE and CBS expression levels induced by DT treatment are functionally related to their reactive sulfur species (RSS) production activity. These RSSs are significantly involved in cytoprotection against oxidative stress. Perhaps the observed adaptive changes in CSE and CBS expression levels upon DT treatment contribute to the protection and survival of melanoma cells against DT-targeted therapy, and thus to the development of drug resistance.

[0080] These findings explain why the rapid induction of other low CSE levels in control cells during DT treatment likely represents an important adaptive response to protect cells during drug exposure. We have concluded that this adaptive response plays a crucial role in building the basis for the survival of drug-resistant cells. CSE is indeed a highly inducible protein, and several transcription factors (see above), including Nrf2, which modulates the antioxidant response in melanoma cells during DT treatment, as well as other stress response factors, have binding sites on the CSE promoter, thereby regulating CSE expression levels (Renga et al., 2009). Therefore, the induction of CSE expression is a rapid adaptive response in cancer, particularly melanoma cells, to counteract cellular damage caused by drug-induced immediate oxidative stress and provide extra fuel for energy production through increased RSS production. When drug resistance develops, CSE levels decrease, elevated CBS levels become dominant, resulting in balanced but increased RSS flow, restored glycolysis, and promotion of cancer cell proliferation and tumor progression.

[0081] Thiosulfates are the final products of the sulfide metabolic pathway and are recognized markers of increased sulfide flow in this field. The inventors found that DT treatment increased the level of thiosulfates (Figure 7C). This result provides further evidence that DT treatment increases the catabolism of sulfides and supersulfides, in addition to increasing the formation of sulfides / supersulfides. Therefore, DT treatment increases sulfide flow.

[0082] Cys levels are generally tightly regulated by oxidative catabolism pathways. In a further surprising finding, despite the high Cys demand in DT-treated cells, DT treatment largely induced oxidative catabolism of Cys. At the protein level, upregulation of the gene for cysteine ​​dioxygenase 1 (CDO1), a Cys metabolic enzyme, was observed in DTR cells. Therefore, oxidative Cys catabolism is likely the main cause of the low steady-state Cys levels in DT-treated and DT-resistant cells.

[0083] In another experiment (data not shown), the ratio of oxidative mutants of protein thiols was found to increase with DT treatment, which provides direct evidence of increased oxidative stress due to DT treatment.

[0084] The metabolite patterns observed herein indicate that standard CBS activity is significantly lower but partially restored in DT-treated control cells compared to controls, which is consistent with measured CBS protein levels. However, standard CSE activity is not prominent in any of these systems, including DT-treated control cells, which were found to be overexpressed. From fraxomics analysis, it can also be concluded that the increased RSS production in DT-treated and DTR cells is not explained by CSE or CBS-mediated metabolism of H2S-producing Cys.

[0085] The inventors also found that DT-treated control cells were characterized by increased oxidative load, both DT-treated control and DT-resistant cells had a high CySSyC:Cys ratio, and that oxidative catabolism of Cys was activated in both DT-treated and DT-resistant cells. This latter observation is particularly surprising in light of the fact that these cells take up more CySSyC and that intracellular GSH, GSSH, and H2S levels are elevated, all of which suggest an increased demand for Cys and resistance to its oxidative catabolism.

[0086] Figure 3F summarizes the major pathways, including the synthesis of GSH, RSS (H2S and cysteine ​​persulfide), and oxidative metabolism to taurine, which, based on our data, are responsible for the increased cys flow in melanoma cells when they are exposed to DT treatment. As described above, the increase in thiosulfate levels due to DT treatment provides further evidence for this effect (see Figure 7C).

[0087] As a further example, the inventors confirmed the above concept through experiments using vemurafenib, the first FDA-approved Braf V600E inhibitor. It was found that a metabolic reprogramming mechanism similar to that of dabrafenib-trametinib acts on the development of V resistance. Consistent with metabolome analysis, the development of vemurafenib resistance in in vitro cell culture proliferation assays was shorter than the development of dabrafenib-trametinib resistance, which is logical as dual inhibition of Braf and MEK results in a more complete blockade of the MAPK / ERK pathway.

[0088] DT treatment also induces metabolic changes confirmed in vivo using xenograft models, and these results are consistent with our in vitro results.

[0089] In a further series of experiments (ETHE1 silencing experiments to improve the effect of DT treatment in A375 cells), the levels of enzymes involved in sulfide catabolism were restored in DT-resistant cells compared to those observed in untreated control cells (Figures 7D-7F).

[0090] All of these, mostly unexpected findings, led the inventors to test CSE inhibition against resistance in this setting. In this test, A375 cells maintained in V-containing medium acquired complete resistance after 2 months, but the proliferation of cells treated with the combination of V and PAG (CSE inhibitor) was significantly reduced (Figure 10A). In the case of DT treatment, resistance development takes approximately 4 months in vitro. However, after 2 months, combination therapy with DT and PAG more effectively inhibited tumor cell proliferation compared to DT treatment alone (Figure 10B). Similar results were obtained with other BrafV600i / MEK1 / 2i, CV, and EB (Figures 10C-10D).

[0091] As another example, the combination of the CSE inhibitor PAG with Braf V600E and MEK1 / 2 inhibitors has been shown to be beneficial in other melanoma cell lines with Braf V600E mutations, such as the SK-Mel28 cell line. In this cell line, it takes longer to acquire acquired resistance than in the A375 cell line, but initial results showed that SK-Mel28 cells receiving PAG in addition to Braf V600E and MEK1 / 2 inhibitors proliferated at a smaller rate.

[0092] The restored level of phospho-MEK1 / 2 is (at least) partially required for acquired resistance. Therefore, we compared phospho-MEK1 / 2 levels in cells cultured with V or DT for 2 months in and without PAG, and importantly, found lower levels of phospho-MEK1 / 2 in PAG-treated cells. Furthermore, phospho-Akt was significantly reduced in cell lines (V, DT, and EB) receiving PAG as a co-treatment, which is important in the development of acquired resistance to BrafV600i / MEK1 / 2i, as shown by us and others. We also confirmed that CSE levels increased upon DT treatment in different Braf V600 mutant cell lines and samples from melanoma patients. Furthermore, they also demonstrated that double administration of DT and PAG delayed the onset of acquired resistance in a mouse model (Figures 11A-C), further strengthening the importance of our invention.

[0093] Another experiment was conducted using CSE knockout melanoma cells to confirm that PAG exerts its effect by inhibiting CSE in overcoming resistance. CSE knockout resulted in a heterogeneous cell population, and although the knockout was successful, a low percentage of cells remained CSE-positive. It was observed that CSE protein levels were virtually undetectable at the start of the experiment (Figure 12A), but after approximately 1.5 months of DT treatment, CSE levels were almost the same in the control cell line and the gene knockout cell line (Figure 12B). Therefore, it can be concluded that cells with incomplete gene knockout proliferate predominantly over knockout cells due to DT selective pressure. This experiment provides further evidence that the process is CSE-specific and therefore CSE-specific inhibitors should be applied to the present invention.

[0094] Therefore, CSE is an important stress response component in MAPK inhibitor therapy and acts as a secondary drug target to enhance the efficacy of BRAF inhibitors and / or MEK inhibitors, such as V and DT targeted therapies.

[0095] BrafV600E mutant melanoma, one of the most deadly forms of skin cancer, remains a significant medical problem because previously introduced, highly effective targeted therapies rapidly become ineffective due to acquired drug resistance. Combined inhibition of Braf and downstream MEK1 / 2 kinases with dabrafenib and trametinib (DT) results in extensive metabolic reprogramming, including increased glutaminolysis and a shift from aerobic glycolysis to oxidative phosphorylation, the latter inducing oxidative stress along with increased CYP activity. This is counteracted by elevated PPP activity and an Nrf2-mediated stress response, resulting in sulfide and hypersulfide production as a result of overexpression of several oxidoreductases, increased GSH, and more cystine uptake, and largely reprogrammed cysteine ​​metabolism. Braf inhibition downregulates CBS and strongly upregulates CSE along with enzymes in the sulfide catabolism pathway, although cells with acquired resistance restored CBS levels and downregulated CSE levels. These findings were consistent with the immediate stress response and the recovery of anabolic pathways in resistant cells upon drug exposure. Using stable isotope tracking, we dissected the enzymatic activity of these two trans-sulfur proteins. Based on our fluxomics data, drug-induced rapid CSE overexpression does not support increased cystic demand but utilizes cystine transported via glutamate-carrying xCT activity to elevate hypersulfide and indirect sulfide levels. Our data indicate that in melanoma cells, hypersulfide / sulfide production induced by BrafV600E inhibition is essential for withstanding drug-induced stress by providing antioxidant protection and a backup electron source for ETC to meet the cell's elevated energy demands. Most importantly, these findings lead to the identification of CSE as a key immediate stress response component in melanoma cells upon drug exposure, which can be utilized in drug development efforts as a secondary drug target to enhance the efficacy of Braf inhibitor therapy.

[0096] Methods for determining BRAF V600 mutations in cancer are well known and readily available to those skilled in the art.

[0097] The BRAF V600 mutation status, for example, the BRAF V600E / K mutation status, can be determined, for example, in stored tissue.

[0098] A well-known method for detecting BRAF V600 mutations is PCR. For example, the cobas® 4800 BRAF V600 mutation test (Roche) is a real-time polymerase chain reaction (PCR) test for the detection and identification of BRAF V600 mutations in formalin-fixed paraffin-embedded tissue (FFPET) of human melanoma (Mourah S. et al., 2015).

[0099] Sanger sequencing is also a suitable method for diagnosing V600 mutations and may provide better results than PCR testing. Combining the two methods can also yield more reliable results (Qu, Kevin et al., 2013).

[0100] It is also preferable that a histological diagnosis of cancer or tumor, such as melanoma, be confirmed by a specialist.

[0101] Treatment methods for V600-mutated cancers or tumors, particularly melanoma, are well-known in this field and are outlined, for example, in the e-book "Systemic therapy options for patients with unresectable melanoma" (Yushak, M. et al. 2017).

[0102] However, in addition to melanoma, the BRAF V600 mutation is also common in several other types of cancer, such as thyroid cancer and non-small cell lung cancer.

[0103] Treatment protocols for treating patients with MAPK inhibitors, particularly BRAF V600 variant inhibitors, and optionally with MEK inhibitors, are well known in the art.

[0104] Examples include, but are not limited to, the treatment of patients with BRAF V600 mutations with dabrafenib and trametinib, which was evaluated in the BELIEVE trial and reviewed by Shimoi T. et al. (Shimoi T. et al., 2024). In these trials, patients with solid tumors were administered dabrafenib (150 mg) twice daily and trametinib (2 mg) once daily until disease progression or unacceptable toxicity was observed. This study confirmed promising efficacy against BRAF V600 mutated tumors and concluded that dabrafenib and trametinib would provide a new treatment option for rare cancers such as high-grade gliomas, biliary tract cancers, and thyroid cancers in patients with BRAF V600 mutations. This conclusion can be extended to other combination therapies with BRAF V600 variant inhibitors, preferably MEK inhibitors.

[0105] As disclosed above, resistance is frequently a problem in such treatments. In a recent review (Florent L. et al. 2023), Florent L. et al. compile recent data on cellular and microenvironment-induced resistance to targeted therapy for BRAF V600 variant metastatic melanoma. While the discovery of targetable mutations and understanding the mechanisms involved in the development of metastatic melanoma have enabled improvements in patient treatment, the authors mention other undiscovered resistance mechanisms and hope that future understanding of such mechanisms may contribute to improved management of melanoma patients. The inventors outline, in particular, the mechanisms involved in resistance to targeted therapy in BRAF V600E variant metastatic melanoma. The treatment methods described in these publications are incorporated herein by reference.

[0106] Treatment with cystathionine-γ-lyase (CSE) inhibitors may fit well with these existing protocols. Treatment of cancers other than those with BRAF V600 mutations has been suggested with CSE inhibitors.

[0107] For example, Liu et al. (Liu et al., 2021) and Wang et al. (Wang et al., 2019) have reported the anticancer effects of CSE inhibition in specific tumors. Li et al. (Li et al., 2021) summarize strategies for controlling breast cancer using inhibitors of H2S-producing enzymes and teach that the CSE inhibitors I194496, I157172, PAG, BCA, and AVG have anticancer activity.

[0108] Typically, the therapeutically effective dose of a CSEi compound can be determined by standard experiments in the art. If an animal model is available, an appropriate dose range for animals can be determined, as well as a set up model (such as the xenograft model with the A375 melanoma cell line prepared by the inventors).

[0109] In one embodiment of the present invention, administration of the compound or pharmaceutical composition of the present invention is initiated at a lower dose, increasing until the desired effect of preventing / treating the relevant medical indication is achieved. This defines the therapeutically effective dose.

[0110] Those skilled in the art, such as clinicians, will be aware of the various factors to consider when determining the optimal dosage of a given subject. Such considerations are known to those skilled in the art. Basically, the FDA Guidelines (Rockville, MD, 2005) can be used. A more detailed discussion of this issue is provided by Nair and Jacob (Nair A.B. and Jacob S., 2016).

[0111] In the case of the CSE inhibitor of the present invention, such human doses, for example, daily doses, are not limited to such doses, but may be, for example, between 0.01 mg / kg and 100 mg / kg body weight, or between 0.05 mg / kg and 50 mg / kg body weight, in particular between 0.1 mg / kg and 10 mg / kg in the case of PAG, or an equivalent dose in the case of another CSE inhibitor.

[0112] The use of pharmaceutical compositions and CSE inhibitors is well known in the art and is disclosed, for example, in U.S. Patent No. 9,725,426 and U.S. Patent No. 1,022,7314, which have been transferred to SOVA Pharmaceuticals.

[0113] Selective inhibitors of cystathionine gamma-lyase (CSE), including PAGs, have long been known and are frequently used examples.

[0114] The selectivity of pharmacological inhibitors commonly used against cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) has been reviewed by Asimakopoulou, A. et al. (Asimakopoulou, A. 2013).

[0115] Li, M et al. (Li, M. et al. 2021) provides an analysis and review of CSE inhibitors, along with specific perspectives on their selectivity. They also identify NSC4056, also known as oulintricarboxylic acid, as the most potent inhibitor with an IC50 of 0.6 μM for up to 2021 CSEs (Hu, Y. et al., 2018). Another compound, 2-allylidenehydrazinecarbodithioate, was found to have higher selectivity for CSEs compared to CBS and to be more active than conventional inhibitors (Bhattacharjee A. et al., 2017).

[0116] example Example 1: Method reagent Unless otherwise specified, all reagents were obtained from Sigma and had a minimum purity of 97%. The Merck organic solvents used for chromatography were gradient grade, deionized with water, and ultrafiltered using an Androna B30 HPLC system. Stable isotopes were prepared by Cambridge Isotope Laboratories. HPE-IAM (β-(4-hydroxyphenyl)ethyliodoacetamide) was purchased from Santa Cruz Biotechnology. Dabrafenib, trametinib, and vemurafenib were obtained from MedChemExpress.

[0117] cell culture A375 cell lines were obtained from Sigma (#88113005) and treated with escalating doses of dabrafenib and trametinib until concentrations of 62.5 nM and 10 nM trametinib were reached, or with vemurafenib until 2 μM was reached. After approximately 3–5 months, cells proliferated in the presence of the inhibitors, were considered resistant, and were further maintained in the presence of the drugs. Cells were maintained in a 5% CO2 incubator at 37°C using 100U penicillin-streptomycin (Lonza#DE17-602E), 2mM L-glutamine (Lonza#17-605E), 10% fetal bovine serum (Sigma#F0392), and high-glucose DMEM (Thermo#21969035) containing 100nM sodium selenite with or without dabrafenib and trametinib or vemurafenib.

[0118] The SK-Mel28 human melanoma cell line was generously provided by the Department of Experimental Pharmacology in Budapest, Hungary, and maintained in a 5% CO2 incubator at 37°C using RPMI1640 medium (RPMI1640, Gibco 11875093) containing 100 U penicillin-streptomycin (Lonza#DE17-602E), 2 mM L-glutamine (Lonza#17-605E), 10% fetal bovine serum (Sigma#F0392), and 100 nM sodium selenite with or without 62.5 nM dabrafenib and 10 nM trametinib. SK-MEL-28 expresses mutant B-Raf (V600E) and wild-type N-Ras, and can form tumors in nude mice, and can be established from patient-derived tumor samples.

[0119] To investigate the effect of PAG on the development of drug resistance, A375 cells and SK-MEL28 cells were cultured in DMEM or RPMI containing 100 nM dabrafenib and 10 nM trametinib, with or without 0.5 mM PAG, as described above.

[0120] Cystine deficiency DMEM (Thermo#21013024) without cystine or methionine was supplemented with 30 mg / L L-methionine, dialysis FBS (Sigma#F0392), 100 U penicillin-streptomycin (Lonza#DE17-602E), 2 mM L-glutamine (Lonza#17-605E), 1 mM sodium pyruvate (Sigma#S8636), and 100 nM sodium selenite. L-cystine was added at different concentrations before the experiment.

[0121] Cell viability and proliferation Cells were seeded in 12 or 24-well plates. After treatment, cells were washed with HBSS, then fixed with cold 10% trichloroacetic acid (TCA), incubated at 4°C for at least 1 hour, and then washed four times with MilliQ water. Sulforhodamine B stain (0.4% in 1% acetic acid) was added to each well and incubated at room temperature for 15 minutes. The plates were rinsed four times with 1% acetic acid and air-dried. 10 mM unbuffered Tris solution was added to solubilize protein-bound SRBs. Absorbance values ​​were measured at 570 nm using a plate reader spectrophotometer.

[0122] Western blot Cells were washed with HBSS and collected in RIPA buffer supplemented with protease and phosphatase inhibitors. After sonication for 15 seconds, cell fragments were removed by centrifugation at 14000 g for 10 minutes at 4°C. Protein concentration was determined by a bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard. Protein samples were denatured in SDS loading buffer and reduced with 100 mM dithiothreitol (DTT) at 95°C for 5 minutes. 15 μg of protein sample per well was loaded onto a polyacrylamide gel. After size separation by electrophoresis, proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo Blotting System (BioRad). Transfer efficiency was verified by Ponceau staining. The membrane was blocked for 1 hour at room temperature with 5% skim milk and 0.5% BSA in TBST (0.05% Tween20).Akt(ab8805), catalase(ab52477), CBS(ab140600), CDO1(ab232699), CSE(ab189916), DLST(ab177934), ETHE1(ab174302), G6PD(ab993) , GAPDH(ab181602), GCLC(ab53179), GPX1(ab108427), GPX4(ab125066), GS(ab124811), GSTpi(ab233112), IDH1(ab172964), Nrf2(ab137 Primary antibodies against 550), OGDH (ab137773), PFKFB3 (ab181861), PFKFB4 (ab137785), SO (ab129094), SOD1 (ab52950), SOD2 (ab13533), Trx (ab185329), and TST (ab166625) are Abcam; CARS2 (HPA041776), MPST (HPA001240), and SQOR (HPA017079) are Sigma; beta-actin (3700S), ERK1 / 2 (4695T), and MEK1 / 2. Antibody (8727), PDH (2784), phospho-(Ser473)-Akt (9271S), phosphor-ERK1 / 2 (Thr202 / Tyr204) (9101L), phospho-MEK1 / 2 (S217 / 221) (9121), and SDHA (5839) were purchased from Cell Signaling; TRP14 (MAB3504) from R&D Systems; GOT1 (MA531527), GOT2 (PA527572), Invitrogen, and TrxR1 (sc-28321) from Santa Cruz Biotechnology. The antibodies were diluted in TBST at a ratio of 1:1000 and incubated with the membrane overnight at 4°C. After washing with TBST (3 × 20 mins), the membrane was incubated with a secondary antibody (anti-mouse / rabbit IgG conjugated with horseradish peroxidase (DAKO)) at a 1:4000 dilution at room temperature for 2 hours. After washing, the membrane was incubated with ECL reagent (BioRad) for 2 minutes, and the signal was detected using a gel documentation system (Syngene).

[0123] RNA isolation and RT-qPCR Cells were cultured in T25 flasks, and total RNA was isolated using TRIzol Reagent (Applied Biosystems) according to the manufacturer's instructions. Concentration and purity were measured spectrophotometrically using a Nanodrop instrument. 3 μg of RNA sample was treated with 2 U of DNase I (ThermoScientific) at 37°C for 30 minutes. 20 mM EDTA was added, followed by thermal inactivation at 70°C for 10 minutes, and the reaction was stopped by immediately placing the sample on ice. The RNA sample was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. The DNA-RNA hybrid product was diluted 10-fold with RNAse-free water and used as a template for real-time quantitative PCR. Analysis was performed on a Roche Light Cycler 480 using 2× qPCRBIO SyGreen Mix reagent (PCRBiosystems) according to the manufacturer's instructions. The relative abundance of mRNA was calculated by ΔCT using GAPDH, β-2-microglobulin (β2M), and actin as invariant controls. The β2M primer was purchased from Qiagen (#PPH01094E). All other primer sequences are listed in Table 1. RT 2 Profiler (trademark) PCR Array Human Cancer Drug Resistance (PAHS-004ZF) and RT 2 The Profiler (trademark) PCR Array Human Cancer Drug Resistance (PAHS-004ZF) was purchased from Qiagen. Regarding the PCR array, RT 2 Using the First Strand Kit (330404), 2 μg of RNA was transcribed according to the manufacturer's instructions and measured using a Roche Light Cycler 480 instrument. [Table 1]

[0124] Preparation of CSE knockout melanoma cell lines Plasmids expressing Cas9 and sgRNA (courtesy of Dr. Mazhar Adli of Northwestern University) were used in all CRISPR experiments. Twenty nucleotide sgRNAs were designed using Benchling.com software. 5'-CACC-3' and 5'-AAAC-3' overhangs were added to the 5' of the forward and reverse complementary oligos, respectively. The forward and reverse sgRNA oligos were mixed in NEBuffer 2 (NEB#B7002S, New England Biolabs), then heated to 95°C and annealed by stepwise reduction, and finally ligated to the BsmBI-cleaved sgRNA expression plasmid before transformation. Positive sequences were confirmed by Sanger sequencing. The vector control included two untargeted 20-nucleotide control guide sequences. HEK293T cells were transfected in OptiMem medium (Gibco #31985070) with target sequences containing plasmids including PsPAX2, Pmd2G, and FuGene6 (Promega E2691) in a 4:1:5 ratio. The virus-containing medium was collected and replaced at 24 and 48 hours, and fresh complete high-glucose DMEM medium was added (see above). The collected medium was then syringe-filtered through a 0.22 μM filter and stored at 4°C for immediate use or at -80°C for long-term storage. Wild-type Cas9-expressing A375 melanoma cell lines were seeded and attached at a concentration of 40-60%. The cells were then infected with a lentiviral mixture containing 8 μg / mL polyblen or control medium, and after 14+ hours, subjected to puromycin (2 μg / mL) selection until all untransfected cells died. After selection, clones were grown from single cells to obtain a homogeneous cell culture. The SgRNA sequences used were as follows: SgCont.: 1.TCATGCTTGCTTGGGCAAAA; SEQ ID NO: 31 2.GCCAGCGGGGATATGGTGAA; SEQ ID NO: 32 SgCSE: 1.TCCAGAGCAATGGACCTCCA; SEQ ID NO: 33 2.AGGCGCCCCTTGCTTGAACG; SEQ ID NO: 34. Competent DH5α E. coli strains were used for all cloning purposes.

[0125] Measurement of mitochondrial activity Using an extracellular Flux Analyzer (Seahorse Biosciences, Seahorse XFp Analyzer), the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured. Cells were seeded in Seahorse culture plates (Seahorse Biosciences, Seahorse XFp FluxPak). For characterization of mitochondrial function, a mitostress test kit (103015) and a glycolysis function glycolysis stress test (103020) were used. After each experiment, the cell culture plates were fixed with 10% TCA and an SRB assay was performed. The OCR and ECAR data were normalized with the measured absorbance data.

[0126] In vivo model In the case of cell line-based xenografts, 10 6 A375 cells in 200 μl of DMEM were subcutaneously injected into 8 - 10-week-old NOD.CB17-Prkdc scid / NCrCrl (NOD-SCID) mice or NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice. The patient-derived tumor xenograft (PDTX) model was donated by KINETO Lab Ltd, Budapest. The model was established from surgically newly removed BRAF V600E mutant treatment-naive melanoma samples in NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA). In the PDTX experiment, tumor samples of approximately 2 × 2 × 2 mm obtained from the fourth generation (G4) of the s.c.-grown model were subcutaneously transplanted into NOD-SCID mice. The width and length of the tumors were measured twice a week with a digital caliper. The volume was estimated using the formula volume = (width) 2 × length / 2. When the tumor reached an estimated 200 mm 3When the tumor volume reached a certain level, mice were randomized and treated a total of four times with 30 mg / kg dabrafenib and 1 mg / kg trametinib / oral. In the case of cell line-derived xenografts, mice were treated daily for four consecutive days. Mice with PDTX were treated on days 1, 4, 5, and 6. Mice were sacrificed 24 hours after the last treatment, the tumor weight was measured, and the tumors were rapidly frozen in liquid nitrogen. The frozen tissue was destroyed using a dismembran.

[0127] For long-term treatment with DT or DT+PAG, 1.5 × 10 6 Individual A375 cells were subcutaneously injected into NSG mice. Tumors were estimated to be 100 mm. 3 Once the tumor volume reached a certain level, mice were randomized and treated orally with 30 mg / kg dabrafenib and 1 mg / kg trametinib five times a week (37 doses in total). In the DT+PAG group, mice were intraperitoneally injected with 5 mg / kg PAG three times a week (23 doses in total). After acquired resistance developed in the DT group, all mice were sacrificed and tumor weight was measured.

[0128] license: KINETO Lab Ltd. holds licenses for animal containment (PEI / 001 / 1715 / 2015) and collection, handling, model generation, and use of PDTX samples (IV / 10147-1 / 2020 / EKU). The provided PDTX cryopreserved samples were transferred to the laboratory animal house of the National Institute of Oncology on dry ice, and the animal containment permit (PEI / 001 / 1738-3 / 2015) and tumor xenograft experiments using anticancer drugs (PE / EA / 1461-7 / 2020) covered all activities during the study. All ethical authorizations were granted by the Hungarian National Committee for Scientific Research Ethics.

[0129] Immunohistochemical analysis of patient samples Formalin-fixed, paraffin-embedded melanoma tumor specimens were collected from the National Institute of Oncology's biobank, and the procedure was approved by the Hungarian National Ethics Committee under file number IV / 10441-1 / 2020 / EKU. After preparing 2 μm thick tissue sections, the sections were deparaffinized and antigen recovery was performed in Roche ULTRA Cell Conditioning (CC1) Solution (pH 6.0) at 95°C for 20 minutes. Anti-CSE antibody (Abcam#ab189916) was used at a 250-fold dilution at 37°C for 20 minutes. Deparaffinization, antigen recovery, and staining were performed using the Roche BenchMark ULTRA IHC / ISH system. Staining was performed using the ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche#760-501) according to the manufacturer's instructions, followed by hematoxylin counterstaining. Images were acquired using an Olympus BX43 microscope equipped with a DP74 camera.

[0130] Lentiviral phenotype introduction For shRNA-mediated stable knockdown of ETHE1, we used MISSION shRNA lentiviral transdermal particles (SHCLNV-NM_014297, ID:TRCN0000083454) at five multiples of infection (MOI). The corresponding control was MISSION pLKO.1-puro empty vector control transdermal particle (#SHC001V). Single clones were grown from the entire population, and ETHE1 expression was evaluated using Western blotting analysis. Clone 9, which had the lowest level of ETHE1, was used for further experiments (Figure 9).

[0131] Isotope tracking experiment For the isotope tracking experiment, the same medium as for cystine deficiency (see above) was used, but either heavy methionine or heavy cystine was added. In the case of heavy methionine labeling, the medium was 30 mg / L L-[ 34The medium contained S]-Met (Cambridge Isotopes#sc-482584A) and 200 μM unlabeled L-cystine (Sigma#C7602). In the case of decystine labeling, the medium contained 30 mg / L unlabeled L-methionine (Sigma number M9625) and 200 μM LU[ 13 C], U[ 15 The medium contained N]-cystine (Cambridge Isotopes no. CNLM-4244-H-PK) (see Figure 3C). After plating and reaching 80% density, the medium was removed, the cells were rinsed with HBSS, and the culture was treated with heavy isotope-containing medium for the time indicated.

[0132] HPLC-MS / MS measurement of amino acids Cells were seeded in 6-well plates, washed with HBSS, and then harvested in CHAPS buffer (150 mM KCl, 50 mM HEPES, pH 7.4, 0.1% CHAPS, protease inhibitor) and sonicated for 10 seconds. After centrifugation (14000 g, 10 min, 4°C), the protein concentration from the supernatant was measured using the BCA method. 50 μl samples with a protein concentration of 1 mg / ml were derivatized with the EZ:Faast kit (Phenomenex, Torrance, CA, USA) according to the manufacturer's instructions and measured using a Thermo Vanquish UHPLC system coupled to a Thermo Q Exactive Focus mass spectrometer. The m / z values ​​recommended by the EZ:Faast kit, supplemented with isotopically labeled versions, were used. The monitored transitions can be found in Table 2. [Table 2]

[0133] HPLC-MS / MS measurement of low molecular weight (LMW) metabolites The measurements were based on the method published by Akaike et al. (Akaike et al., 2017), as described herein: Cells were seeded in 6-well plates, washed twice with HBSS, and harvested in ice-cold methanol containing 5 mM β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM). For measurements from xenograft tumors, approximately 10–40 mg of freeze-ground tissue samples were homogenized in 5 mM HPE-IAM. Samples were kept on ice between each preparation step. After sonication, alkylation was performed at 37°C for 20 minutes, followed by centrifugation (14000 g, 10 min, 4°C). The supernatant was acidified with 10% formic acid and diluted 2-fold with 0.1% FA / H2O before injection. Cell pellets were dissolved in 1% SDS / PBS, sonicated, and protein content was measured using a BCA assay. HPLC-MS / MS measurements were performed using a Thermo Q-Exactive Focus Orbitrap mass spectrometer connected to a Thermo Vanquish UHPLC. Samples were measured using two different methods.

[0134] To measure extracellular metabolites, 50 μl of culture medium was alkylated with 5 mM HPE-IAM at 37°C for 20 minutes. The sample was acidified with 10% TCA and centrifuged at 14000 g for 10 minutes at 4°C.

[0135] The first method was performed using eluents 0.1% FA / H2O (A) and 0.1% FA / MeOH (B) on a Phenomenex Kinetex C18 (50 × 2.1 mm, 2.6 μm) column. The initial 5% B was linearly increased to 13% in 2 minutes, then to 95% in 4 minutes, held for 0.5 minutes, then returned to 5% B in 0.1 minutes, and held for 3.4 minutes before the next injection. The flow rate was 0.5 ml / min at 40°C. MS / MS detection was performed in positive ionization mode, and the analytes listed in Table 2 were detected using high-energy collision dissociation (HCD).

[0136] A Phenomenex Hypercarb (100 × 2.1 mm, 3 μm) column was used in the second technique, with eluents of 0.5% FA / H2O (A) and 0.5% IPAIN 1:1 (B). The initial 0% B increased linearly to 30% in 15 minutes, then increased to 100% in 1 minute, held for 5 minutes, then decreased to 100% A in 1 minute, held for 8 minutes. The temperature was 40°C and the flow rate was 0.2 ml / min. The analytes were detected by MS / MS in positive ionization mode using high-energy collision dissociation (HCD).

[0137] HPLC-MS / MS measurement of taurine and hypotaurine Cells were seeded in 6-well plates, washed twice with HBSS, and harvested in ice-cold 75% methanol solution. For measurements from xenograft tumors, approximately 10–40 mg of freeze-ground tissue samples were homogenized in ice-cold methanol. Samples were kept on ice between each preparation step. After sonication, precipitated proteins were removed by centrifugation (14000 g, 10 min, 4°C). The supernatant was acidified with 10% formic acid (FA) and diluted 2-fold with 0.1% FA / ACN before injection. Cell pellets were dissolved in 1% SDS / PBS, sonicated, and protein content was measured using a BCA assay. HPLC-MS / MS measurements were performed using a Thermo Vanquish UHPLC and a Thermo Scientific LTQ-XL mass spectrometer coupled to a Phenomenex Kinetex HILIC column (100 × 2.1 mm, 2.6 μm) according to previously published methods.

[0138] HPLC-MS / MS measurement of persulfurized proteins The measurements were performed as described herein, based on the method published by Akaike et al. (Akaike et al., 2017): Cells were seeded in a 6-well plate, washed with HBSS, collected with 5 mM HPE-IAM in RIPA buffer, and sonicated. After centrifugation (14000 g, 10 min, room temperature), 100 μl of supernatant was desalted using a Zeba spin column (7K MWCO, 0.5 mL). Protein content was measured from the flow-through using the BCA method, and then 100 mM HPE-IAM in DMSO was added to the desalted solution. The protein levels of the desalted samples were equalized using RIPA buffer and digested at 37°C for 1 hour with pronase (3 mg / ml) in 35 mM sodium acetate buffer (pH 5.0). Undigested proteins were precipitated by adding 10% TCA, and the samples were centrifuged (14000 g, 10 min, room temperature). The supernatant was injected into an LC-MS / MS, and the derivatized analytes were measured using the same HPLC-MS / MS method described above for LMW species, with a Kinetex C18 column. MS / MS detection was performed in positive ionization mode, and cysteine ​​and cysteine ​​persulfide were detected using high-energy collision dissociation (HCD).

[0139] Measurement of thiosulfate (S2O3) using an alkylation protocol based on monobromobimane Cells were seeded in 12-well plates and washed once with PBS. After adding 100 μl of PBS pH 8.0 containing 1 mM monobromobiman, the cells were scraped off, collected, and incubated at 37°C for 1 hour to label the thiol groups. The reaction was stopped using 10 μl of 50% TCA, and the precipitated protein was removed by centrifugation at 4000 g at room temperature for 5 minutes. The protein was then redissolved in 4% SDS containing 0.1 M NaOH for the BCA protein assay.

[0140] Derivative detection from the supernatant was performed using a Thermo Ultimate 3000 HPLC system equipped with a fluorescence detector. 5 μl of the derivatized sample was injected into a Phenomenex Kinetex XB-C18 150x3 mm 2.6 μm column and separated by gradient elution using 0.1% TFA / H2O (A) and 0.1% TFA / MeOH (B) as follows: The flow rate was set to 0.6 ml / min with an initial composition of 10% B. After 3 minutes, a linear increase was introduced with 15% B for 9 minutes. Subsequently, the column was washed with 75% B for 2 minutes, equilibrated with 10% B for 3 minutes, and then the next injection was performed.

[0141] Monobromoximan-labeled hydrogen sulfide was measured from the same sample using different chromatographic methods with 0.1% TFA / H2O (A) and 0.1% TFA / ACN (B) on a Phenomenex Luna C18(2) 250x2mm 2.6um column using the following gradient elution: The flow rate was set to 0.25 ml / min, and the initial composition of 15% B was increased to 35% B in 3 minutes. At 8.5 minutes, another linear increase was introduced to 90% B in 2 minutes. 90% B was held for 1 minute, returned to 15% B in 1 minute, and after further equilibration at 15% B for 2 minutes, the next injection was performed.

[0142] For fluorescence detection of the labeled analyte, an excitation wavelength of 390 nm and an emission wavelength of 475 nm were selected, and quantification was performed by establishing a calibration curve prepared from standard solutions.

[0143] Example 2 2.1. In addition to activating the PI3K / Akt pathway, dabrafenib-trametinib-resistant cells overexpress ABC transporters and cytochrome P450 proteins. To investigate the molecular and metabolic background of melanoma resistant to MAPK inhibitors (MAPKi), we created a DT-resistant line of BRAF V600E mutant A375 human melanoma cells (A375-DTR) by long-term culture in the presence of escalating doses of dabrafenib (BRAF inhibitor) and trametinib (MEK1 / 2 inhibitor) (Figure 1A). The viability of resistant cells in the presence of the drugs was tracked using a proliferation assay (Figure 1B). Decreased activity of the MAPK / ERK pathway was confirmed by measuring phosphorylated (active) MEK1 / 2 and ERK1 / 2 (Figure 1C). After 48 hours of treatment of control (A375-control) cells with DT, MEK1 / 2 phosphorylation was inhibited, but DT-resistant cells partially recovered MEK1 / 2 phosphorylation. Furthermore, inhibition of the MAPK-ERK pathway resulted in increased Akt phosphorylation and activation (Figure 1D), which was consistent with previous reports demonstrating that increased activity of the PI3K-Akt pathway contributes to DT resistance (Liu et al., 2020; Sun et al., 2014). Using Qiagen's real-time quantitative PCR (RT-qPCR) based array, we identified overexpression of several additional genes involved in the development of cancer drug resistance (Figures 1E, 1F). We confirmed the increased expression of these genes using custom-designed oligonucleotides (Figure 1E). In addition to increased expression of the ATPase H+ transport V0 subunit d2 (ATP6V0D2), apolipoprotein E (APOE), heat shock protein B1 (HSPB1), and ATP-binding cassette subfamily G member 2 (ABCG2) gene (Figure 1G), which are generally involved in the neutralization and efflux of xenobiotics, the inventors found overexpression of cytochrome P450 family (CYP) members CYP1B1, CYP2C13, CYP2F1, and CYP17A1 (Figure 1G). CYP proteins play diverse roles in cellular metabolism by oxidizing steroids, fatty acids, drugs, and xenobiotics. As CYP is a heme protein and acts as a terminal oxidase, it is an important player in the production of endogenous reactive oxygen species (ROS) (Veith and Moorthy, 2018).

[0144] 2.2. Changes in the redox environment in DT-treated and DT-resistant (DTR) melanoma cells The inventors created dabrafenib and trametinib-resistant (DTR) A375 cells (Figures 1A-C) and examined the expression of drug resistance-related genes (Figures 1D, 1G, and 8A, 8B). In particular, they found overexpression of a member of the Cytochrome P450 (CYP) family (Figure 1E), which produces reactive oxygen species (ROS) while neutralizing xenobiotics (Veith and Moorthy, 2018). Overexpression of CYP enzymes may contribute to the previous observation that A375 cells resistant to Braf and MEK inhibitors show increased ROS levels (Corazao-Rozas et al., 2013; Wang et al., 2018). Overexpression of CYP enzymes may contribute to increased oxidative flow observed in Braf inhibitor (BRAFi)-resistant A375 cells, in addition to heated oxidative phosphorylation (Corazao-Rozas et al., 2016; Corazao-Rozas et al., 2013; Wang et al., 2018). Furthermore, increased OXPHOS in DT-treated and DT-resistant cells may also contribute to increased ROS production (see below). To counteract oxidative stress, (BRAFi)-resistant melanoma cells exhibit strong activation of the nuclear factor erythroid factor 2-related factor 2 (Nrf2) transcription factor, resulting in activation of the pentose phosphate pathway (PPP) and increased xCT expression (Khamari et al., 2018). Here, we found very high levels of Nrf2 in DT-treated cells and confirmed a slight accumulation of Nrf2 in DT-resistant cells (Figure 1F). Therefore, we confirmed the accumulation of Nrf2 in DTR cells and showed that it decreased upon drug deficiency, and found that Nrf2 levels further increased significantly over 7 days in DT-treated control cells. Thus, the Nrf2-mediated antioxidant response was immediately after DT exposure and remained partially active in DTR cells (Figure 1F).This protein expression pattern was found to be similar for catalase (CAT), superoxide dismutase 2 (SOD2), and several important antioxidant proteins, including members of the thioredoxin (Trx) and glutathione (GSH) systems, such as thioredoxin reductase 1 (TrxR1), 14kDa human thioredoxin (Trx)-related protein (TRP14), and glutathione peroxidases 1 and 4 (GPX1, 4), suggesting that DT-induced changes in antioxidant gene expression levels are primarily mediated by Nrf2 (Figure 1G). TrxR1 was unique among these enzymes, similarly showing an exceptional elevation in DTR cells.

[0145] In addition to elevated intracellular glutathione levels, the inventors found that enzymes involved in ROS neutralization were overexpressed not only in resistant cells but also in DT-treated cells (Figure 1H). High levels of the peroxide and superoxide scavenging enzymes catalase (CAT) and superoxide dismutase 2 (SOD2) were observed in DT-treated controls and DT-resistant cells compared to untreated controls, and gradually decreased in DT-deficient resistant cells. The same trends were measured for members of the thioredoxin (Trx) and glutathione (GSH) systems, such as thioredoxin reductase 1 (TrxR1), 14kDa human thioredoxin (Trx)-related protein (TRP14), and glutathione peroxidases 1 and 4 (GPX1, 4). Notably, TrxR1 was unique among these enzymes by being exceptionally elevated in DT-resistant cells. Enzymes in the Trx and GSH systems generally play a role in maintaining cellular redox homeostasis through sequential redox reactions to protein and peptide cysteine ​​residues (Lu and Holmgren, 2014). The central hubs of the two systems are TrxR1 and glutathione reductase (GR), which convert the cellular reducing power derived from nicotinamide adenine dinucleotide phosphate (NADPH) into the reduction of oxidative Cys modifications. The majority of NADPH is produced by the pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis. Glucose-6-phosphate dehydrogenase (G6PD), an enzyme in the PPP, is involved in the reduction of NADP + It is involved in the conversion of to NADPH, and by measuring its levels, the inventors observed the same trend in a melanoma cell model as in the case of previous oxidoreductases (Figure 1I), further confirming the high need for these cells for increased antioxidant capacity.

[0146] The majority of the reducing power utilized by the Trx and GSH systems originates from nicotinamide adenine dinucleotide phosphate (NADPH), which is primarily produced by the PPP enzyme glucose-6-phosphate dehydrogenase (G6PD). Consistent with the expected antioxidant capacity requirements of treated melanoma cells, G6PD expression levels in our model showed a similar trend to the aforementioned oxidoreductases (Figure 1G).

[0147] During glucose metabolism, the flow between glycolysis and PPP is regulated by members of the bifunctional 6-phosphofructose-2-kinase / fructose-2,6-bisphosphatase (PFKFB) family. The kinase activity of PFKFB3 diverts glucose towards glycolysis, while the fructose-bisphosphotase activity of PFKFB4 redirects glucose back towards PPP (Yi et al., 2019). These enzymes are attracting increasing attention in cancer biology. For example, PFKFB4 depletion inhibited tumor growth in prostate cancer cells in a xenograft model by enabling a catastrophic accumulation of ROS (Ros et al., 2012). Furthermore, we previously showed that PFKFB4 expression was elevated in CBS-silencing breast cancer cells, potentially associated with increased oxidative stress in these cells (Erdelyi et al., 2021). By measuring the protein levels of PFKFB3 and PFKFB4, we found that PFKFB3 levels decreased and PFKFB4 levels increased in DT-treated control cells, consistent with redirected glucose flow to PPP to support increased NADPH demand in cells during DT treatment (Figure 1H). On the other hand, in DT-resistant (DTR) cells, levels of both PFKFB3 and PFKFB4 were almost restored to the levels observed in untreated control cells, which, along with the antioxidant protein expression profile, suggests a more balanced redox environment and reactivation of the glycolytic pathway (Figure 1J).

[0148] In summary, the increase in oxidative stress upon inhibition of the MAPK / ERK pathway in melanoma cells by DT treatment is counteracted by Nrf2-mediated overexpression of several antioxidant genes. This antioxidant response is supported by a transition from glycolysis to PPP and a corresponding increase in NADPH production to fuel the antioxidant mechanism. In DT-resistant cells, a more balanced redox environment and restoration of glycolysis are predicted based on the expression patterns of enzymes that regulate the cellular antioxidant response and glucose metabolism.

[0149] 2.3. Cellular energy metabolism is reformed in DT-treated cells and DT-resistant cells. Nrf2 (see above), which is elevated in DT-treated and DTR cells, is also involved in redirecting glucose and glutamine anabolic pathways (Mitsuishi et al., 2012). Furthermore, it is widely accepted that BRAF mutations can reprogram cellular metabolism; for example, V600E mutant BRAF maintains glycolytic activity, and therefore addiction to glycolysis effectively becomes addiction to BRAF V600E itself (Hall et al., 2013). Thus, BRAF inhibitors significantly alter metabolism in melanoma cells. It has already been shown in other groups that melanomas exposed to BRAFi rapidly become dependent on oxidative phosphorylation (OXPHOS) for survival, as demonstrated by the high sensitivity of BRAFi-treated cells to apoptosis induction in response to several mitochondrial respiratory chain inhibitors (Corazao-Rozas et al., 2016; Corazao-Rozas et al., 2013). Therefore, the inventors measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a cell model system using the Seahorse Cell Analyzer and found that DT-resistant cells have higher basal respiration, but both DT-treated and DT-resistant cells have higher non-mitochondrial oxygen consumption, maximal respiration, and higher respiratory reserve capacity (Figure 2A). The inventors also investigated the glycolytic activity of these cells and found that DT-treated control cells have lower glycolytic activity, volume, and reserve capacity, but these are (partially) restored in DT-resistant cells (Figure 2B). A fundamental feature of cancer cell metabolism is increased aerobic glycolysis to support macromolecular synthesis in order to meet the metabolic demands of rapidly proliferating cells (DeBerardinis and Chandel, 2016). Therefore, it is not surprising that resistant cells require restored glycolytic activity to restore their ability to proliferate. This is consistent with a more balanced redox environment and less need for excessive NADPH production by PPP as described above, which allows cells to restore their anabolic mechanisms.

[0150] Consistent with the antioxidant response, increased mitochondrial respiration and decreased glycolytic activity, accompanied by increased levels of citrate cycle proteins, were observed in DT-treated control cells to balance drug-induced oxidative stress (Figure 2C) (Corazao-Rozas et al., 2016; Corazao-Rozas et al., 2013). In DTR cells, we found increased mitochondrial respiration, increased levels of pyruvate dehydrogenase (PDH) and succinate dehydrogenase (SDHA), and restored glycolytic activity consistent with a balanced redox environment, enabling both the restoration of anabolic mechanisms and cell proliferation (Figure 2C) (DeBerardinis and Chandel, 2016).

[0151] Next, the inventors measured the protein levels of citrate cycle (CAC) enzymes involved in providing electron donors for electron transport chains (ETCs) such as NADH. The inventors found very high levels of pyruvate dehydrogenase (PDH), oxoglutarate dehydrogenase (OGDH), and dihydrolipoamide S-succinyltransferase (DLST), which are responsible for NADH production, in DT-treated controls and moderately high levels in DTR cells. Succinate dehydrogenase (SDHA), which is not only a CAC enzyme but also a member of the ETC and is responsible for the reduction of coenzyme Q, was also very elevated in DT-treated cells, but its level did not decrease, and was rather even higher in DTR cells (Figure 2C).

[0152] This enzyme pattern is consistent with the increased flux through ETC in the presence of DT treatment to support the increased energy demands of cells.

[0153] Next, the inventors performed mass spectrometry metabolome analysis to measure intracellular and extracellular amino acid levels. Targeted amino acid metabolome analysis revealed increased levels of intracellular glutamine (Gln), asparagine (Asn), and aspartic acid (Asp) in DT-treated control cells, and a slight increase in Asp in DT-resistant cells (Figure 2D). Furthermore, both DT-treated control cells and DTR cells took up more Gln (Figure 2E), which is consistent with previous observations that the Nrf2 pathway is upregulated in these cells, as Nrf2 also promotes Gln consumption (Mitsuishi et al., 2012). Interestingly, the inventors found increased extracellular levels of Asn in the culture medium of DT-treated control cells and DTR cells, as well as increased levels of Asp in DTR cells (Figure 2E), suggesting that the metabolic pathways involved in the conversion of these amino acids are also specifically reprogrammed. Therefore, the inventors investigated the expression of genes involved in glutamate (Glu) metabolism. Glu is a non-essential amino acid and plays a central role in antioxidant defense, metabolic reprogramming, and oncogenic signaling in cancer cells (Zhu and Thompson, 2019). In relation to our previous observations, Glu can be converted to α-ketoglutarate (α-KG) to supply CAC, which is a building block in GSH synthesis and is used by the xCT antiporter to transport cystine into the intracellular matrix. Glutamine synthetase (GLUL) converts Glu to Gln, while glutaminase 1,2 (GLS1,2) enzymes synthesize Glu from Gln by glutaminolysis (Figure 2F).DT-treated control cells were found to upregulate the expression of GLS1 and GLS2 genes, while resistant cells were found to upregulate GLS1 expression and strongly suppress GLUL expression (Figure 2G). This suggests that DT-treated cells upregulate glutaminolysis to satisfy their extra need for Glu, while DT-resistant cells suppress the conversion of Glu to Gln to supply the increased Glu demand, consistent with increased Gln uptake, as shown above and in previous studies (Figure 2G) (Baenke et al., 2016; Khamari et al., 2018). Previous studies have also suggested that Nrf2-mediated redirection of glucose and glutamine metabolism in MAPK inhibitor-resistant cells (Baenke et al., 2016; Khamari et al., 2018), despite the use of V-resistant cells, correlates with our findings. Glutamate oxaloacetate transaminases 1 (GOT1, cytoplasm) and 2 (GOT2, mitochondria) promote the reaction between aspartate and α-ketoglutarate, producing glutamate and oxaloacetate (Recasens et al., 1980). They are members of the malate-aspartate shuttle and therefore play an important role in metabolite exchange between mitochondria and the cytoplasm, amino acid metabolism, and the regulation of intracellular NAD(H) redox balance. However, no significant difference in their expression levels was observed in this cell model system (Figure 2H).

[0154] In summary, the data presented herein suggest that metabolic pathways involved in cellular energy are deeply reprogrammed in BrafV600Ei and MAPKi-resistant melanoma cells. This includes upregulation of OXPHOS accompanied by increased expression of the CAC enzyme, which produces NADH that fuels the electron transport chain (ETC). Along with the upregulation of these pathways, we found increased glutaminolysis in DT-treated and DT-resistant cells, which indicates an increased demand for Glu.

[0155] It has been previously reported that MAPK inhibitor-resistant melanoma cells are more vulnerable to transcriptional inhibition of xCT countertransporters involved in cystine (CySSyC) uptake via extracellular transport of Glu (Wang et al., 2018). Consistent with this concept, we found increased sensitivity of DT-resistant (DTR) cells to CySSyC deficiency-induced ferroptosis cell death, but only in the absence of selenite in the culture medium (Figure 3A). Since selenocysteine-containing enzymes (selenoproteins) require selenite for selenocysteine ​​synthesis, for consistency, we supplemented the cell culture medium with 100 nM sodium selenite unless otherwise indicated. The reason behind the fact that DTR cells were more susceptible to CySSyC deficiency in the absence of selenite supplementation (compared to controls) may be related to the adaptive overexpression of the selenium protein GPX4 in both cell lines (see Figure 1I), which is one of the major protective factors against ferroptotic cell death (Dixon et al., 2012). Indeed, when cells were cultured in a medium without excess selenite, GPX4 levels (along with two other major selenium enzymes, TrxR1 and GPX1) decreased significantly within 24 hours, making the cells more vulnerable to CySSyC deficiency (Figure 3B). Consistent with this observation and previous reports (Khamari et al., 2018; Wang et al., 2018), we found increased CySSyC uptake by DT-resistant cells by measuring the decrease in CySSyC levels and the increase in Glu levels in the culture medium on the cells. Furthermore, the inventors found that DT-treated control cells also showed increased CySSyC uptake (Figure 3C). This is consistent with the inventors' previous observation that increased Glu production by glutaminolysis is present in both DT-resistant and DT-treated cells, supporting (among other factors) increased xCT antiporter activity. The inventors also measured increased CySSyC influx via xCT in control cells as well as DTR cells (Figure 3C), which is consistent with increased Glu production by glutaminolysis in both cell lines (Figures 2D-G).The elevated intracellular glutathione levels observed in DTR cells (Figure 3D) (Wang et al., 2018) are likely to be utilized not only in their redox buffering capacity (particularly through the supply of GPX-catalyzed reactions) but also to provide more substrates for the cytoprotective and xenobiotic neutralizing activities of the glutathione-S-transferase (GST) enzyme. Indeed, along with elevated GSH levels, we also found increased expression of the most prominent xenobiotic catabolic GST isoform, GST pi, in DT-treated and DT-resistant (DTR) cells (Figure 3E).

[0156] In summary, the data presented herein highlight that DT-treated cells and DT-resistant cells require increased levels of CySSyC to counteract oxidative stress and promote GSH synthesis, which is utilized to neutralize anticancer drugs.

[0157] 2.4. MAPK inhibitors reprogram cys metabolism Next, we investigated how increased uptake of CySSyC in DT-treated and DT-resistant (DTR) cells alters Cys metabolism. After entering the cell, CySSyC is readily reduced to Cys in the cytoplasmic environment by the Trx system (Pader et al., 2014). DTR cells require more Cys to feed their increased GSH production and therefore certainly contribute to increased CySSyC uptake. However, Cys is not only utilized for GSH synthesis; other Cys utilizing cellular pathways can produce taurine, hydrogen sulfide, or Cys-SSH (Figure 3F). Interestingly, despite increased CySSyC uptake, we found lower intracellular Cys levels in DT-treated and DT-resistant cells compared to untreated controls (Figure 3G), suggesting higher Cys flow through its metabolic events. Furthermore, the inventors found that intracellular levels of CySSyC were 2.5 times higher in DT-treated control cells than in untreated cells, and the CySSyC / Cys ratio was significantly higher in DT-treated controls and DT-resistant cells compared to untreated controls, demonstrating a transition to an oxidized state. These observations are consistent with increased oxidative stress and increased CySSyC uptake in the presence of the BrafV600E inhibitor. Cys uptake occurs mainly via xCT in its oxidized form (CySSCy), but it can also be synthesized from methionine (Met) via the trans-sulfurization pathway and can therefore be considered a semi-essential amino acid. In this process, homocysteine ​​(HCys) is produced from Met through multiple steps, followed by the conversion of HCys to cystathionine (CTH) by cystathionine β-synthase (CBS) (Figure 4B, reaction 1), which is then utilized for Cys production by cystathionine γ-lyase (CSE) (Figure 4B, reaction 2) (Kumar and Banerjee, 2021; Sbodio et al., 2019). The inventors then investigated whether elevated Cys is required during DT treatment and whether the sensitivity of resistant cells to CySSyC deficiency is accompanied by an altered pattern of Cys producing trans-sulfurases.Inhibition of the MAPK / Erk pathway by DT in control cells was found to induce rapid and significant expression of CSE accompanied by a decrease in CBS levels (Figure 3I / 1). Interestingly, the opposite pattern was observed in DT-resistant (DTR) cells, with restored CBS expression and a decrease in CSE to near-undetectable levels (Figure 3I / 1). Upregulation of CSE by 5 days of DT treatment was also observed in another Braf V600 mutant cell line, SK-MEL28 (Figure 3I / 2). Since intracellular synthesis of Cys requires the sequential action of CBS and CSE, these enzymatic patterns do not support the idea that trans-sulfurization is reprogrammed upon DT treatment to supply the cell's elevated Cys demand. However, both CBS and CSE can independently generate reactive sulfur species (RSS) simultaneously via the reverse trans-sulfurization pathway (Ida et al., 2014; Yadav et al., 2016). Indeed, the increased Cys-SSH / Cys ratio in DT-treated control cells, the elevation of H2S2 in DTR cells, and the higher concentrations of H2S and GSSH in both DT-treated and DTR cells (Figures 3G and J) suggest that the changes in CSE and CBS expression levels induced by DT treatment are functionally related to their RSS production activity. In addition to CSE and CBS, another enzyme, mercaptopyruvate sulfur transferase (MPST), is also involved in H2S production (Ida et al., 2014; Modis et al., 2013; Nandi et al., 2000) and Cys persulfide conversion (Pedre and Dick, 2021; Pedre et al., 2023). We found a slight increase in cytoplasmic MPST (upper band) in DT-treated control cells (Figure 3I / 1), suggesting a possible contribution of MPST to the increase in RSS levels observed in these cells (Figures 3G and J). However, MPST-mediated RSS production requires 3-mercaptopyruvate (3MP), which is produced from Cys by the GOT enzyme.Given that Asp is a preferred substrate for GOT over Cys, in DT-treated controls and DT-resistant cells where Asp levels were elevated (see Figure 2D) and no increase in GOT expression was observed (see Figure 2H), GOT-mediated 3MP production, and therefore MPST-mediated sulfide production, is unlikely to be the primary cause of the observed increase in RSS levels (Pedre and Dick, 2021; Ubuka et al., 1992).

[0158] RSS plays a significant role in cytoprotection against ferroptosis and other oxidative stresses (Barayeu et al., 2022; Doka et al., 2020; Zivanovic et al., 2019), supplying energy to ETC (Akaike et al., 2017; Hanna et al., 2022; Libiad et al., 2019; Szabo et al., 2014), and regulating aerobic glycolysis (Vitvitsky et al., 2021). Therefore, we propose that the observed adaptive changes in CSE and CBS expression levels during DT treatment are likely to contribute to the protection and survival of melanoma cells against DT-targeted therapy, and consequently to the development of drug resistance.

[0159] Panza et al. previously showed that A375 cells are sensitive to sulfur donors because these proteins downregulate both the MAPK / Erk and PI3K / Akt pathways, and that overexpression of CSE in A375 cells inhibits cell proliferation (Panza et al., 2015). Furthermore, Leikam et al. showed that knockdown or pharmacological inhibition of CSE in A375 cells reduces proliferation and leads to senescence (Leikam et al., 2014). These findings explain why CSE levels are low in control and proliferating DTR cells, and that its rapid induction in control cells upon DT treatment likely represents an important adaptive response to protect cells during drug exposure. This adaptive response undoubtedly plays a crucial role in building the survival base of drug-resistant cells. CSE is, in fact, a highly inducible protein regulated by a wide range of stimuli, including oxidative stress. Several transcription factors, including Nrf2, which modulates the antioxidant response in melanoma cells during DT treatment (Sbodio et al., 2019), as well as other stress response factors SP1 and ATF4, have binding sites on the CSE promoter and thereby regulate CSE expression levels (Renga et al., 2009).

[0160] In summary, our data, in light of previous observations, suggest that induction of CSE expression (Figure 3I) is a rapid adaptive response by melanoma cells to counteract cell damage caused by drug-induced immediate oxidative stress (see Figures 1F-1H) and to provide extra fuel for energy production through increased RSS production (Figures 2A-2B) (Figure 3J). When drug resistance develops, CSE levels decrease, and increased CBS levels (Figure 3I) take over, resulting in balanced but increased RSS flow (Figure 3J), which restores glycolysis (Figures 2A-B), promoting cancer cell proliferation and tumor progression, as in many tumor types, and demonstrating that elevated CBS contributes to tumor progression (Ascencao and Szabo, 2022; Coletta et al., 2012; Czikora et al., 2022; Erdelyi et al., 2021).

[0161] This observation may explain why CSE levels are lower in control and proliferating DTR cells, and why its rapid induction in control cells upon DT treatment likely represents an adaptive response to drug exposure. CSE is, in fact, a highly inducible protein regulated by a wide range of stimuli, including oxidative stress (Sbodio et al., 2019).

[0162] Intracellular Cys levels are strictly regulated by an oxidative catabolic pathway mediated by cysteine ​​dioxygenase (CDO), which converts Cys to cysteinesulfinic acid (CSA), which is then decarboxylated by cysteinesulfinic acid decarboxylase (CSAD) to produce hypotaurine and taurine, or deaminated to release β-sulfinylpyruvate. CDO is highly sensitive to steady-state levels of Cys (Stipanuk and Ueki, 2011), and CDO concentrations can increase up to 45-fold in some cells upon excessive Cys exposure (Dominy et al., 2006). To the great surprise of the inventors, despite the high Cys requirements of DT-treated cells and the low intracellular steady-state Cys concentrations, they found that DT treatment significantly induces oxidative catabolism of Cys. The inventors measured a 20-fold increase in CDO expression at the mRNA level in DT-resistant cells, and a 7-fold and 1.5-fold increase in downstream cysteine ​​sulfinate decarboxylase (CSAD) enzyme in DT-treated cells and DTR cells, respectively (Figure 3H). The increased expression of CDO1 in DTR cells was confirmed at the protein level by Western blotting (Figure 3I). These findings suggest that oxidative cyscatalysis is likely the main cause of the low steady-state cys levels in DT-treated cells and DT-resistant cells.

[0163] Figure 3F summarizes the major pathways, including the synthesis of GSH, RSS (H2S and cysteine ​​persulfide), and oxidative metabolism to taurine, which, based on our data, are responsible for the increased cys flow in melanoma cells when they are exposed to DT treatment.

[0164] 2.5. Insights into how Cys metabolism is reprogrammed in A375 cells by a fraxomic approach using stable isotopes. To gain deeper insights into how the Cys metabolic pathway is rearranged during DT treatment in melanoma cells, and to explain the above observations, we performed a comprehensive targeted metabolome analysis of the trans-sulfurization pathway.

[0165] First, the inventors measured the total levels of Cys metabolic intermediates in the absence of heavy isotopes and found that the total levels of HCys, CTH, and Lanth decreased in DT-treated control cells, but were accompanied by a significant increase in Hlanth levels (Figure 4A). CTH levels were almost eliminated in DT-treated control cells, considering the standard Cys production pathway, which is consistent with the fact that CTH-producing CBS levels were low and CTH-consuming CSE levels were high, suggesting that the non-standard activity (despite being elevated) of CSE producing CTH and H2S from Cys and HCys is negligible (Figure 4E, reaction VI). On the other hand, since Hlanth can only be produced from two HCys cells by CSE (Figure 4E, reaction VII), the increase in Hlanth levels along with the decrease in HCys cells suggests that this reaction may contribute to some extent to the increase in sulfide levels observed in DT-treated controls (Figure 3J). Lanth is another metabolite and may exhibit CSE or CBS catalytic production of the H2S reaction using two Cys molecules as substrates (Figure 4E, reaction V). However, Lanth levels were lower in DT-treated control cells, meaning these reactions cannot explain the observed increase in RSS. In DT-resistant cells, CTH and Lanth levels were still significantly lower than in untreated control cells but higher than in DT-treated control cells, reflecting partially restored CBS activity compared to DT-treated control cells, but indicating that the increase in RSS levels in DT-treated and DTR cells is not generated by the CBS / CSE catalytic conversion of Cys and HCys to CTH and H2S (Figure 4E, reaction VI) or the conversion of 2Cys to Lanth and H2S (Figure 4E, reaction V).

[0166] Next, the inventors measured the flow through metabolic pathways using Met or CySSyC isotopes in cultures in culture media (when stable Met or CySSyC isotopes were used in their culture media, the normal and heavy LMW thiol metabolic profiles of the trans-sulfur pathway were measured in their cell model systems). First, to investigate the canonical functions of CSE and CBS, the inventors used heavy sulfur containing Met (Met*) ( 34Cells were treated with S). ​​An 18-hour treatment was sufficient to completely replace the Met and HCys pools with heavy Met and HCys. Under these conditions, we found that Cys synthesis from Met was negligible in all systems tested, even after 48 hours of cell proliferation (Figure 4C), which is consistent with the abundant supply of CySSyC available to cells from the culture medium and the differential expression patterns of CBS and CSE (Figure 3I). By measuring the ratio of heavy CTH to total, we found that in untreated control and DT-resistant cells, approximately 60% of CTH originated from the canonical pathway (Figure 4B, reaction 1), compared to only 30% in DT-treated control cells. These data support our previous findings that DT-treated control cells overexpress CSE and downregulate CBS (Figure 3I), so they cannot synthesize the same amount of CTH from HCys via CBS action as untreated control cells (Figure 4B, reaction 1). However, since this reaction is catalyzed not only by CBS but also by CSE, CTH can be synthesized from Cys (Figure 4F, reaction 8) (shown as light CTH in this experiment). Importantly, since the production of CTH from Cys also produces H2S (Figure 4F, reaction 8), these data suggest that increased CSE-induced metabolism of Cys to CTH may contribute to increased RSS production in DT-treated control cells. However, this is not supported by the fact that in DT-treated control cells, heavy HCys levels (not their ratio to the total of heavy and light analytes as described above) are lower compared to control cells, and heavy CTH is almost completely eliminated, whereas in DT-resistant (DTR) cells, these are restored to control levels (Figure 4D). This latter observation is explained by the fact that DT-resistant cells have restored levels of CBS and low levels of CSE compared to untreated control cells (Figure 3I), and are able to synthesize CTH from HCys and Ser (via CBS) (Figure 4B reaction 1), but have a deficiency in converting this CTH to Cys (due to low CSE) (Figure 4B reaction 2). These observations are also consistent with data and conclusions drawn based on total metabolite measurements in the absence of isotopic labeling (see above and Figure 4A).

[0167] In summary, these metabolite patterns indicate that standard CBS activity is extremely low in DT-treated control cells but partially restored in DTR cells compared to controls, consistent with measured CBS protein levels (Figure 3I). However, standard CSE activity is not prominent in either of these systems, including DT-treated control cells, and is even overexpressed (Figure 3I). It can also be concluded that the increased RSS production in DT-treated and DTR cells is not explained by CSE or CBS-mediated metabolism of H2S-producing Cys.

[0168] In the following experiment, the inventors added heavy carbon ( 13 C) and heavy nitrogen ( 15Using CySSyC containing N), the levels of normal and heavy (*) analytes were measured to better understand the RSS production function of CSE and CBS in these cell lines. By calculating the ratio of heavy analytes to total levels, the inventors found that after 18 hours of treatment, the intracellular CySSyC and Cys pool was completely replaced with heavy CySSyC and Cys (Figure 4G). Next, the inventors calculated the incorporation of carbon skeletons derived from CySSyC (not from Met on the canonical pathway) into CTH, Lanth, and Ser by comparing the heavy analytes to their corresponding total levels. The inventors found that under normal cell culture conditions (200 μM CySSyC), Lanth and CTH production from Cys is substantial, but Ser synthesis from Cys is negligible (Figure 4G). By analyzing the steady-state levels of heavy metabolites (and their ratio to the sum of light and heavy metabolites), we found that untreated control cells synthesized more CTH (Figure 4F reaction 8) and Lanth (Figure 4F reaction 9) from heavy Cys than DT-treated or DT-resistant cells (Figure 4G), which is consistent with our previous results that untreated control cells have higher intracellular concentrations of Cys, the substrate in these reactions (Figure 3G), as well as higher total Lanth and CTH levels (Figure 4A). These results also support the conclusion that reactions V and VI (Figure 4E) do not contribute to the elevation of RSS in DT-treated and DTR cells.

[0169] Ida et al. demonstrated that CySSyC can also be directly used by CBS and CSE to produce CySSH (Ida et al., 2014), which can sulfide other Cys derivatives via a persulfurization reaction (Ida et al.), and increase the sulfide concentration via the action of the Trx or GSH system (Doka et al., 2020; Wedmann et al., 2016). However, kinetic simulations by the Banerjee group suggested that under physiological conditions, this pathway is not prominent due to low intracellular CySSyC concentrations and high Cys levels, and that an increase in the CySSy to Cys ratio is necessary for it to play a role in CySSH synthesis (Yadav et al.). (al., 2016). As the inventors have shown above, DT-treated control cells are characterized by increased oxidative load, and both DT-treated control and DT-resistant cells show an increased CySSyC to Cys ratio. Furthermore, the inventors found that oxidative catabolism of Cys is activated in DT-treated cells and DTR cells (Figure 3H, Figure 3I). This latter observation seemed almost counterintuitive, given that these cells take up more CySSyC (Figure 3C) and intracellular GSH, GSSH, and H2S levels are elevated (Figure 3J). All of these suggest an increased demand for Cys to counteract its oxidative catabolism. However, utilization of Cys by CSE or CBS is not shown in DT-exposed cells. The following is a viable mechanism that can reconcile this anomaly with the fact that it cannot explain the observed increased levels of S: Increased oxidative cyscatalysis and oxidative stress, along with increased uptake of CySSCy in DT-treated and DTR cells, increases the CySSyC to CyS ratio to some extent, thereby making CySSCy a prominent substrate for CBS and / or CSE, enabling direct production of Cys-SSH under these conditions. It must also be recognized that CySSCy is approximately two orders of magnitude better than Cys as a substrate for these enzymes, and that the fact that this reaction is not supported under normal conditions is due to a reduced atmosphere in which much of the cytoplasm is reduced (Ida et al., 2014).

[0170] Nevertheless, higher steady-state levels of H2S and GSSH in DT-treated and DTR cells could protect them from oxidative stress-induced ferroptosis (Barayeu et al., 2022; Wu et al., 2022) or metalloprotein-induced oxidative stress (Doman et al.). Furthermore, persulfidation of Cys residues can reversibly reduce oxidatively modified persulfides (perthiosulfen / sulfin / sulfonic acid) back to thiols, thus maintaining protein function under oxidative stress (Doka et al., 2020; Filipovic et al., 2018). Therefore, we measured the total levels of high molecular weight (HMW) sulfides to evaluate whether DT-induced RSS elevation can protect protein Cys residues from drug-induced oxidative stress (to investigate the potential of this thiol protection mechanism against drug-induced oxidative damage). In fact, the inventors found elevated levels of protein-Cys-SSH and protein-Cys-SSSH in DT-treated control cells after 6 days of treatment, but observed no significant difference in protein persulfidation between untreated control resistant cells and DT-resistant cells (Figure 4I). This may be related to the fact that DT-treated control cells need to cope with unexpected oxidative stress (having the highest levels of oxidative stress), and may induce increased persulfidation of protein Cys residues via a rapid CSE-mediated response to protect the most oxidatively sensitive protein thiols before the cells can adapt to the stress conditions.

[0171] As described herein, Braf V600E and MEK1 / 2 inhibition using dabrafenib and trametinib (DT) upregulates enzymes in the sulfide catabolism pathway. This pathway is involved in sulfide clearance, donating electrons to the electron transport chain and thus regulating mitochondrial energy metabolism.

[0172] In a further series of experiments, the increased activity of this pathway upon DT treatment was confirmed by measuring the final product, thiosulfate (S2O3), using a monobromobimane-based alkylation protocol, followed by fluorescence detection. A375 cells were treated as described above.

[0173] Previous findings indicate that DT treatment increases the activity of the sulfide catabolism pathway, potentially donating electrons to the electron transport chain, but this activity is restored to normal in DT-resistant A375 cells treated with dabrafenib-trametinib (DT) (Figure 7C).

[0174] It should be noted here that the enzymes in the sulfide degradation pathway clearly show the same pattern. Their levels correlate with the levels of the final product of the pathway (thiosulfate).

[0175] In these experiments, DT is also administered to resistant cells to maintain their resistant phenotype. This is due to the nature of cell-level experiments, as cells can easily alter their phenotype in an altered environment. In these experiments, we are interested in what DT does to control cells (acute effect) and how it differs from resistant cells that receive DT but can divide in the same way as controls. In these and other experiments, such as those measuring mitochondrial function, CSE / CBS levels, resistant cells show a similar pattern to untreated controls, even though DT is present throughout the culture medium.

[0176] 2.6. Inhibition of BrafV600E by vemurafenib results in similar redox changes in melanoma cells. Vemurafenib was the first FDA-approved Braf V600E inhibitor (Figure 5A), but tumors rapidly developed acquired resistance to this drug, primarily due to downstream hyperactivation of MEK kinase (Manzano et al., 2016; Robert et al., 2015), which resulted in combined inhibition of the MAPK / ERK pathway, a common practice in medical oncology (see the combination of dabrafenib and trametinib). However, to determine whether current observations regarding Cys and H2S metabolic rewiring are a common adaptive response in melanoma cells to Braf V600E inhibition, we investigated how Cys and H2S metabolism changes in vemurafenib (V)-treated cells and vemurafenib-resistant (VR) cells. First, we created vemurafenib-resistant A375 cell lines by long-term culture of cells in the presence of escalating doses of vemurafenib. Cell proliferation was investigated by an SRB assay, confirming that VR cells had fully recovered their proliferative capacity (Figure 5B). By measuring MEK1 / 2 and ERK1 / 2 phosphorylation, it was found that V treatment effectively blocked MEK1 / 2 phosphorylation, and that MEK1 / 2 phosphorylation was fully restored in VR cells. Next, in the case of DT treatment, etc., the inventors measured the levels of proteins involved in cellular defense against oxidative stress and / or Cys metabolism (Figure 5C). Similar to DT treatment, the inventors found that V treatment in control cells resulted in reduced levels of CBS, but CSE, MPST, PDH, G6PD, TrxR1, and GPX1, 4 proteins were overexpressed. Furthermore, in V-resistant cells, these enzyme levels recovered to mostly untreated control levels, similar to what the inventors observed in the case of DTR cells. However, TrxR1 levels also recovered in VR cells, in contrast to DTR cells, and we detected even higher TrxR1 concentrations in DTR cells compared to DT-treated control cells. Consistent with previous results, vemurafenib treatment in control cells induced a decrease in steady-state Cys, CTH, and HCys levels and increased CySSyC, H2S, GSSH, and Hlanth levels.On the other hand, in VR cells, the inventors found significant differences only in Cys-SSH levels and CTH levels. Interestingly, in VR cells, Cys, CySSyC, H2S, HCys, and Lanth levels were fully restored, and in fact, CTH levels were even higher than in untreated control cells (Figure 5D). These data suggest that a similar metabolic reprogramming mechanism to that the inventors found for DT resistance functions in the development of V resistance, but the observed differences caused by DT treatment are more pronounced, indicating that resistance and metabolic balance to V treatment developed more rapidly. Consistent with metabolome analysis, the onset of vemurafenib resistance in the in vitro cell culture proliferation assay was faster than the onset of dabrafenib-trametinib resistance, which is not surprising since dual inhibition of Braf and MEK results in complete blockade of the MAPK / ERK pathway.

[0177] 2.7. Using a xenograft model and during V therapy of cells, DT-induced metabolic changes during BRAF inhibition were confirmed in vivo. To investigate metabolic changes in melanoma tissue during DT treatment in vivo, the inventors established two xenograft mouse models using (1) subcutaneous injection of A375 cells (A375-X) and (2) transplantation of patient-derived tumor samples into immunodeficient mice (PDX). Dual treatment of mice with dabrafenib and trametinib resulted in a rapid reduction in tumor volume (Figures 6A and 6C), demonstrating the high efficacy of this targeted therapy. To gain insight into metabolic differences in DT-treated tumors, the inventors performed metabolome analysis on both A375 cell line-derived xenografts (A375-X) and patient-derived xenograft (PDX) tumor samples. In both models, a rapid reduction in tumor volume indicated a high initial response rate to DT treatment (Figures 6A and 6C). In DT-treated A375-X tumors, tissue metabolome analysis showed a decrease in steady-state levels of Cys, CTH, and Lanth, and an increase in persulfide formation of Cys and GSH, while increases in CySSyC and GSSH levels were observed in accordance with our in vitro results. Furthermore, we found that in DT-treated A375-X tumors, Cys and GSH species shifted to their oxidized dimerized state (Figure 6B). In DT-treated PDX tumors, we also found a decrease in steady-state levels of Cys, CTH, and Lanth, but increased steady-state levels of CySSyC, Hlanth, and Cys-SSH. The shift to oxidized forms of Cys and GSH during DT treatment (more pronounced oxidation of Cys and GSH) was also significant in PDX tissue (Figure 6D). These together provided in vivo evidence for our in vitro findings (Figure 6D). As in vitro, in both tumor models, the greatest effect of DT treatment was on CTH, by reducing the level of this metabolite by more than 90% compared to that measured in untreated tumor tissue. This, along with the observed low steady-state Cys levels, means that the increase in RSS levels in DT-treated tumors is not due to the reverse H2S-producing trans-sulfurization activity of CSE and / or CBS using Cys as a substrate (Figure 4E, Reaction VI).Our in vitro fraxomics results showed that, under sufficient CySSyC availability, the standard Cys production function of CBS and CSE is negligible, while their sulfide / hypersulfide production function is more prominent. However, in xenograft tumors, nutrients are not equally available to all tumor cells, and nutrient availability depends on angiogenesis in the tumor tissue. In tumor cells with insufficient CySSyC availability, the Cys production function of CBS and CSE may also be essential for survival. Gaining deeper insights into these details would require considerable effort and an in vivo fraxomics approach, which is outside the scope of this study. Furthermore, in mouse xenografts, detection of human proteins is also quite difficult because mouse tissue infiltrates human tumors, and the protein sequences of human and mouse CBS and CSE show high sequence homology, limiting the specificity of available antibodies and making their differentiation by shotgun proteomics almost impossible. Therefore, these in vivo metabolome analyses are in complete agreement with the results obtained in our cell model system.

[0178] Furthermore, the inventors found that another BrafV600E inhibitor, vemurafenib(V) (which was the first FDA-approved drug for treating Braf-mutant melanoma patients), induced similar changes in Cys metabolism in melanoma cells. Importantly, V-treated cells also showed downregulation of CBS and overexpression of CSE accompanied by increased RSS production (Figure 5).

[0179] 2.8. ETHE1 silencing improves the effect of DT treatment in A375 cells. Since intracellular sulfide levels are strictly regulated by catabolic pathways, and Cys-persulfide species also supply these mechanisms (Akaike et al., 2017; Combi et al., 2023; Fujii et al., 2019; Marutani et al., 2021), the inventors investigated the levels of proteins involved in sulfide catabolism, such as quinone oxidizer (SQOR) that oxidizes sulfide:H2S to GSSH, persulfide dioxygenase (ETHE1 or PDO) that catalyzes the oxidation of GSSH to yield sulfites and GSH, thiosulfate sulfur transferase (TST) that oxidizes GSSH and sulfites to thiosulfates, and sulfite oxidase (SO) that is involved in sulfite clearance. The inventors found that the levels of all four enzymes were strongly elevated in DT- and V-treated control cells (Figures 7A and 7B, respectively), which, along with the increase in CSE levels, suggested a greater flow of RSS through melanoma cells upon BrafV600E inhibition. High H2S concentrations inhibit mitochondrial respiration (complex IV), but at lower concentrations, electrons can be donated to ETC via SQOR and CoQ to stimulate mitochondrial respiration (Goubern et al., 2007; Szabo et al., 2014). Thus, SQOR can not only detoxify sulfides but also stimulate mitochondrial respiration. Furthermore, recent studies have suggested that LMW persulfide is also a potent electron donor for ETC (Akaike et al., 2017; Fujii et al., 2019). These results, along with the inventors' mitochondrial energy experiments (see above), suggest that increased intracellular RSS flow is involved in supplying the greater energy demands of melanoma cells upon BrafV600E inhibition. The levels of enzymes involved in sulfide catabolism were restored in DT-resistant cells to those observed in untreated control cells. This is also consistent with all of our previous data suggesting that cells are under less stress at the onset of resistance, which restores their anabolic mechanisms with reduced OXPHOS and restored glycolysis (Figure 1J, 2A-B).It has been suggested that high levels of H2S can also uncouple mitochondrial respiration and promote reverse electron transport (RET) associated with increased ROS production, either through inhibition of complex IV or due to an excessively reduced CoQ pool (Banerjee and Kumar, 2022; Jia et al., 2020; Kumar et al., 2022). In DT-treated and DTR cells, only a slight (1.5-fold) increase in endogenously produced H2S levels was detected, and previous studies have suggested that BrafV600E inhibitor-treated melanoma cells are particularly sensitive to respiratory chain inhibitors (Corazao-Rozas et al., 2016), which together means that an increase in endogenous sulfide levels in this system does indeed promote mitochondrial respiration. However, the possibility that a lack of electron acceptors may induce RET to some extent under these conditions cannot be ruled out.

[0180] In summary, our data suggest that increased intracellular RSS flow is not only important in protection against oxidative stress, but also related to the observed higher electron flow through ETC to supply the increased energy demand of melanoma cells when BrafV600E inhibition (see also Section 2.3) is required to activate transporters and efflux DT, e.g., ABCG2 (see Figure 1G). In DTR cells, sulfide catabolic enzymes were restored to untreated control levels, which is consistent with the previously proposed idea that cells were not stressed at the time of resistance development (Figures 1G-1I, 3G), and that their anabolic mechanisms, OXPHOS and glycolysis, which had been reduced, were restored (Figures 2A-B).

[0181] To further investigate the role of mitochondrial RSS in the development of DT resistance, the inventors established the lentiviral ETHE1 silencing A375 cell line from a single clone (Figure 7D). The inventors found no significant changes in the expression of Cys and H2S metabolic pathway enzymes during ETHE1 silencing (Figure 7E). However, when cells were treated with DT, shETHE1 cells showed lower levels of SQOR, SO, and TST, and higher levels of TrxR1 compared to sh control cells. Furthermore, the inventors found increased TrxR1 expression in DT-treated shETHE1 cells compared to DT-treated sh controls. Since the Trx system is important for reducing hypersulfide and polysulfide levels (Doka et al., 2016), overexpression of TrxR1 may be a compensatory mechanism in Ethe1 silencing cells to reduce the level of sulfide escaping into the cytoplasm. Overexpression of TrxR1 may be due to the adaptation of ETHE1-silencing cells to increased hypersulfide levels in the cytoplasm (Doka et al., 2016). We also investigated the proliferation of these cells and, interestingly, found that shETHE1 cells proliferated slightly slower than untreated control cells in a 1-week in vitro proliferation assay (Figure 7E). However, when cells were treated with DT, the proliferation of shETHE1 cells was much more pronounced in a 3-week in vitro proliferation assay, demonstrating that low levels of ETHE1 are preferable for A375 cells when treated with DT, thus implying the importance of sulfide in cell survival during DT treatment (Figure 7F).

[0182] Next, LMW thiols and GSSH (substrates of ETHE1) were also measured in sh control and shETHE1 cell lysates (Figure 7G). GSSH is the most abundant LMW persulfide in the cells, and under normal conditions it is oxidized to sulfite and GSH by ETHE1. The inventors compared untreated shETHE1, DT-treated control, and DT-treated shETHE1 cells with untreated sh control, and also compared DT-treated shETHE1 cells with DT-treated sh control cells to elucidate differences that may contribute to increased resistance to DT treatment. Compared with untreated sh control cells, the inventors found higher levels of GSH, gGluCys, GSSH, and GSSG in DT-treated shETHE1 cells, but lower levels of CTH were measured in both DT-treated sh control and shETHE1 cells. Surprisingly, lower levels of Lanth were observed in shETHE1 cells but not in DT-treated shETHE1 cells. Compared to DT-treated control cells, DT-treated ETHE1 cells showed high levels of Cys, GSH, gGluCys, and GSSG, but low intracellular levels of CySSyC. We also measured LMW thiols from the culture medium and found that GSSH levels increased up to 3-fold in the medium above that of DT-treated ETHE1 cells compared to all other cells (Figure 7H). Collectively, the increase in intracellular levels of GSSH is accompanied by increased levels of GSH, GSSG, and gGluCys, a precursor of GSH synthesis. Our data suggest that since ETHE1 silencing cells are impaired in recovering GSH from GSSH, these cells may need to enhance their intracellular GSH synthesis mechanisms to maintain GSH homeostasis for Gpx1-mediated antioxidant defense, GSTpi-mediated drug metabolism, and Gpx4-mediated protection from ferroptosis (see above). Furthermore, if ETHE1 cannot utilize the excess GSSH generated during DT treatment, the cells will expel it into the extracellular space.Although the inventors did not find a significant increase in the total level of protein persulfide in DT-resistant cells (see above), they found higher levels of protein persulfation in DT-treated controls and shETHE1 cells, particularly DT-treated shETHE1, compared to untreated sh controls (Figure 7I). These observations are consistent with the mechanism proposed by the inventors, in which CSE-produced persulfide also provides protein thiol protection during DT-induced oxidative stress.

[0183] 2.9. CSE is overexpressed in melanoma patient samples upon DT treatment. To evaluate the physiological reliability of the therapeutic potential of combined inhibition of CSE with BRAF V600E targeted therapy, the inventors measured CSE expression levels in patient samples after DT treatment. Collecting clinical samples from patients receiving targeted therapy at the time of sampling is extremely difficult, as surgical removal of melanoma is performed only in rare scenarios when the treatment remains effective. In the biobank of the Hungarian National Institute of Oncology, which treats the largest number of melanoma patients in Europe, the inventors found only one patient for whom pre- and post-treatment cutaneous melanoma samples were available. Immunohistochemistry clearly demonstrated a strong increase in CSE levels in tumor samples removed during DT treatment (Figure 8A). Across patient-derived samples, elevated CSE expression (magenta staining) was generally observed in metastatic patient samples. However, the inventors found another patient with paired pre- and post-treatment samples from lymph node metastases, where CSE levels were high in metastases, but even higher levels of CSE were observed in lymph nodes, which were removed during DT treatment (Figure 8B). By comparing lymphoid melanoma metastases from different patients, the inventors found significantly lower CSE levels in four out of five untreated tumors and higher CSE levels in one out of five compared to patients treated with DT (Figure 8C). These findings strongly suggest that CSE is upregulated in melanomas actually exposed to DT treatment, and strengthen the idea that this upregulation is an important stress response mechanism during BRAF V600E inhibition.

[0184] 2.10. CSE inhibition inhibits the development of resistance to BrafV600E inhibitors. As detailed above, our results show that DT or V-treated A375 cells had elevated RSS levels (Figures 3J and 5D), and silencing persulfide dioxygenase increased the growth rate of these cells in a 3-week growth assay (Figure 6D). Inhibition of BrafV600E and MEK did not kill all melanoma cells in vitro, with some remaining quiescent. We have accumulated extensive data indicating that reprogrammed Cys and RSS metabolism plays a major role in protecting these cells against BrafV600E and Mek inhibitors, as well as against drug-induced oxidative stress. We have demonstrated that the immediate adaptive response in this system is an increase in CSE levels and their sulfide / hypersulfide production activity to protect oxidation-sensitive protein thiols and supply the cells' increased energy demand by providing electrons to ETC (Figures 3I-J). To evaluate whether increased CSE expression during targeted therapy can act as an Achilles' heel for persistent melanoma cells, we used selective CSE inhibitors in the following experiments. Importantly, we found that treatment-naive A375 melanoma cells were insensitive to both the selective CSE inhibitor PAG (Figure 9A) and the non-selective CSE inhibitor AOAA (Figure 9B). This can be inferred from the fact that CSE is lower in treatment-naive melanoma cells and upregulated by targeted therapy. In the following experiments, we used the CSE inhibitor PAG and treated cells with V, CV, DT, or EB in the presence and absence of PAG to evaluate how important the CSE-induced stress response is in the survival and development of drug resistance in melanoma cells during BrafV600E and MEK inhibition. A375 cells were maintained for 2 months in V or DT-containing medium with or without PAG, and these cells were used for a 7-day proliferation assay. The inventors found that A375 cells maintained in a culture medium containing only V acquired complete resistance after two months, while the proliferation of cells maintained in the presence of both V and PAG was significantly impaired (Figure 10A). Furthermore, drug resistance was evident after two months of treatment of cells with DT, but to a low degree (resistance development takes approximately four months in vitro).However, when DT was used in combination with PAG, the development of resistance was prevented. After two months, combination therapy with DT and PAG more effectively inhibited tumor cell proliferation compared to DT treatment alone (Figure 10B). In the case of CV treatment, a significant portion of the cells died due to CV and CV+PAG treatment. After 10 weeks of treatment with CV±PAG, CV-treated cells partially recovered their proliferative capacity, while CV+PAG-treated cells remained quiescent (Figure 10D). In the case of EB treatment, which also induces CSE overexpression as with V, CV, and DT, EB+PAG treatment was more effective than EB alone after four months (Figure 10C). Several different mechanisms may be involved in this phenomenon. As we have previously shown, CSE / CBS / MPST or CARS2-induced persulfide formation plays a crucial role in several processes involved in cancer cell survival, including angiogenesis, protection against hypoxia and ferroptosis (Coletta et al., 2012; Czikora et al., 2022; Erdelyi et al., 2021; Szabo, 2016).

[0185] The restored levels of phospho-MEK1 / 2 are (at least) partially necessary for acquired resistance; therefore, to explore specific effects in this system, we compared phospho-MEK1 / 2 levels in cells cultured with V or DT for 2 months in the presence and absence of PAG, and with EB for 4 months in the presence and absence of PAG. Importantly, we found lower levels of phospho-MEK1 (low band) in PAG+V-treated cells than in V-treated cells (Figure 10E) and EB+PAG (Figure 10G) compared to EB, suggesting that CSE-induced RSS may promote MEK1 phosphorylation in the presence of the V600E inhibitor and contribute to the development of drug resistance (see Figures 10E and F). Interestingly, MEK2 (upper band) phosphorylation was stronger in both V+PAG and DT+PAG-treated cells than in cells treated with V or DT alone, a point that we cannot explain. The PI3K / Akt pathway is often upregulated in Braf / MEK inhibitor-resistant human melanoma (Sun et al., 2014), and consistent with this, increased Akt phosphorylation was observed in our system as well (see Figure 1D). Importantly, we found lower levels of phospho-(Ser473)-Akt in cells cultured with Braf and / or MEK inhibitors in combination with PAG compared to when the drugs were used alone (Figures 10E-10H). This may be related to CSE-induced persulfurization of MEK1, which has been proposed to induce ERK phosphorylation and activation (Zhao et al., 2014) and could (at least partially) explain the decreased cell proliferation observed in the presence of PAG (Figures 10A-10D).

[0186] Our in vitro data support that in A375 cells, CSE is a key stress response component during DT treatment and acts as a secondary drug target to enhance the efficacy of V, CV, DT, and EB targeted therapies. Next, to clarify whether combined inhibition of CSE and BRAF / MEK is beneficial under in vivo conditions, we established a xenograft model derived from the A375 cell line in NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG) mice. Tumor-bearing mice were treated with either DT or a combination of DT and PAG, and both treatments effectively reduced tumor growth (Figure 11A). In the DT-only group, the first tumor exceeding the size measured before treatment occurred at day 24 of treatment, whereas in the DT+PAG group, this occurred at day 38 (Figure 11A). In summary, after 25 days of treatment, tumors in DT-treated mice developed acquired resistance, but an increase in mean tumor volume in DT+PAG-treated mice was observed only at approximately 20 days (Figure 11A). Treatment was considered effective until the tumor reached the volume measured before treatment initiation (progression-free survival, PFS). PFS in DT+PAG-treated mice was significantly higher than in mice treated with DT alone (Figure 11B). At 50 days of treatment, PFS was evident in 9 out of 11 mice in the DT+PAG arm, while only 1 out of 12 mice in the DT-only arm remained in a PFS state. After sacrificing the mice and removing the tumors, tumor weight was significantly lower in the DT+PAG group when measured 50 days after the initial treatment (Figure 11C).

[0187] These results indicate that inhibition of CSE effectively delays the onset of acquired resistance to BrafV600 inhibitors and MEK1 / 2 inhibitors under in vivo conditions.

[0188] Example 3 3. Combined inhibition of the MAPK / ERK pathway and CSE may delay the onset of acquired resistance in the SK-Mel28 cell line. To demonstrate that combining the CSE inhibitor D-,L-propargylglycine (PAG) with Braf V600E inhibitors and MEK1 / 2 inhibitors is beneficial in other melanoma cell lines carrying the Braf V600E mutation, we began culturing the SK-Mel28 cell line with Braf V600E and MEK1 / 2 inhibitors, with or without PAG. In the case of this cell line, it takes longer to acquire acquired resistance than the previously used A375 cell line, but our preliminary results show that SK-Mel28 cells receiving PAG in addition to Braf V600E and MEK1 / 2 inhibitors already proliferate at a slower rate.

[0189] Example 4 4. DT treatment leads to greater proliferation of CSE-expressing cells than CSE-deficient cells in the polyclonal cell population. To demonstrate that the beneficial effect of PAG in delaying acquired resistance is clearly attributable to the crucial role of CSE in the survival of persistent cells, we constructed a stable CSE knockout A375 cell line using CRISPR-Cas9 technology (see example). In general, CSE knockout was successful, and CSE levels were barely detectable in the CSE knockout cell population (Figure 12A).

[0190] However, this cell population is polyclonal, and CSE knockout may not have been successful in all cells. After 1.5 months of DT treatment, we used Western blotting to find no difference in CSE protein levels between the control and CSE knockout cell populations (Figure 12B), which means that cells expressing CSE proliferated more than cells in which CSE knockout was successful. This further reinforces our previous observation that CSE is indeed crucial in the development of acquired resistance, and that its inhibition is a viable approach to inhibit or delay the onset of acquired resistance.

[0191] Example 5 5.1 Exemplary diagnosis and treatment of patients with the BRAF V600E mutation A patient with a dark nevus that had been found to have increased in size over the past two months visited our outpatient clinic. A biopsy was performed on the discovered dark nevus, and after analysis, it was diagnosed as malignant melanoma.

[0192] Further analysis by the treating physician is required to determine whether the melanoma is a BRAF V600 melanoma.

[0193] Patients are instructed to be treated with combination dabrafenib-trametinib therapy, which is completed with PAG therapy where each drug is applied in a standard treatment regimen.

[0194] 5.2 Exemplary diagnosis and treatment of patients with the BRAF V600E mutation Patients with cutaneous melanoma with high metastatic potential are receiving vemurafenib treatment and experience tumor regression after 3 months of treatment. However, recurrence is observed at the 6-month follow-up visit.

[0195] A biopsy is taken and sent to the treating physician for tissue analysis to determine if the melanoma is a BRAF V600 melanoma. The melanoma is then diagnosed as a BRAF V600K variant.

[0196] The patient's treatment regimen was updated to include trametinib and PAG, with the latter at twice the dose compared to that described in Example 5.1. [Industrial applicability]

[0197] The present invention is particularly useful for preventing or delaying the onset or development of resistance in patients to the treatment of Braf V600 mutant cancers, particularly melanoma, using MAPK inhibitors, especially inhibitors of Braf V600 variants. The present invention also relates to combination therapies, which may optionally involve diagnosis, as well as pharmaceutical compositions, combinations, and kits for such therapies.

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Claims

1. A cystathionine-γ-lyase (CSE) inhibitor for use in combination with one or more MAPK inhibitors to treat BRAF V600 mutation-positive cancer in patients, for use in preventing or delaying the onset or development of acquired resistance in patients to cancer treatment with one or more MAPK inhibitors, The MAPK inhibitor includes a BRAF V600 variant inhibitor. Cystathionine-γ-lyase (CSE) inhibitor.

2. The CSE inhibitor for use according to claim 1, wherein the CSE inhibitor is selective for CSE.

3. The cancer is a BRAF V600 mutation-positive melanoma, and the MAPK inhibitor includes a BRAF V600 inhibitor. Here, preferably, the mutation in the BRAF V600 mutant inhibitor is selected from the group consisting of V600D, V600K, V600R, or V600E, more preferably V600K or V600E, and more preferably V600E. A CSE inhibitor for use according to any one of claims 1 and 2, preferably for use in preventing or delaying the development of acquired resistance in the patient to the treatment of the cancer with one or more MAPK inhibitors, wherein the patient is a mammalian patient, preferably a human patient.

4. The MAPK inhibitor includes a BRAF inhibitor, preferably a MEK inhibitor. A CSE inhibitor for use according to claim 3.

5. A CSE inhibitor for use according to any one of claims 1 to 4, for use in preventing or delaying the onset of acquired resistance in a patient to a tumor, wherein the tumor is a BRAF V600 mutation-positive cancer having a BRAF V600D, V600K, V600R, or V600E mutation.

6. A CSE inhibitor for use according to any one of claims 3 to 5, wherein the BRAF inhibitor is selected from vemurafenib, dabrafenib, or encorafenib.

7. A CSE inhibitor for use according to any one of claims 3 to 6, wherein the MEK inhibitor is selected from trametinib, cobimetinib, binimetinib, or selumetinib, more preferably selected from trametinib, cobimetinib, or binimetinib, and more preferably the MEK inhibitor is trametinib.

8. The CSE inhibitor for use according to any one of claims 3 to 7, wherein the MAPK inhibitor comprises a BRAF V600 inhibitor selected from vemurafenib, dabrafenib, and encorafenib, and a MEK inhibitor selected from cobimetinib, trametinib, and binimetinib, preferably the combination of the BRAF inhibitor and the MEK inhibitor is dabrafenib + trametinib, vemurafenib + cobimetinib, or encorafenib + binimetinib, and more preferably dabrafenib + trametinib.

9. A CSE-specific CSE inhibitor for use according to any one of claims 3 to 8, for use in preventing or delaying the onset of acquired resistance to BRAF V600E inhibitors and MEK inhibitors in BRAF V600E mutant cancers.

10. The CSE inhibitor is selected from propargylglycine (PAG), β-cyanoalanine (BCA), L-aminoethoxyvinylglycine (AVG), hydroxylamine, I194496, I157172, S-3-carboxypropyl-L-cysteine ​​(CPC), NSC4056 (orintricarboxylic acid), L-aminoethoxyvinylglycine, 2-allylidene-hydrazinecarboditioate, or cystathionine-γ-lyase-IN-1 (CAS number 2165706-30-7). Preferably, a CSE inhibitor for use according to claim 9, selected from β-cyanoalanine (BCA), L-aminoethoxyvinylglycine (AVG), and propargylglycine (PAG), more preferably D,L-propargylglycine (2-aminopenta-4-ic acid or H-DL-Pra-OH) or N-propargylglycine (2-propyne-1-ylamino)acetic acid), and very preferably propargylglycine (PAG).

11. A CSE inhibitor for use according to any one of claims 1 to 10, wherein the onset of resistance is delayed by at least one, two, three, four, five, six months or more.

12. The CSE inhibitor is administered before the administration of the MAPK inhibitor, or The CSE inhibitor is administered simultaneously with the MAPK inhibitor, or The CSE inhibitor is administered after the administration of the MAPK inhibitor, or the CSE inhibitor and the MAPK inhibitor are administered in sequential, intermittent, or continuous therapy. A CSE inhibitor for use according to any one of claims 1 to 11.

13. A pharmaceutical kit comprising a cystathionine-γ-lyase (CSE) inhibitor and a MAPK inhibitor for use in the treatment of a patient's BRAF V600 mutation-positive cancer, in combination with (one or more) MAPK inhibitors, preferably for use in preventing or delaying the onset / development of resistance to the treatment of the patient's cancer with the MAPK inhibitor, A pharmaceutical kit comprising the aforementioned MAPK inhibitor, which includes a BRAF V600 variant inhibitor.

14. A pharmaceutical composition comprising a cystathionine-γ-lyase (CSE) inhibitor and a MAPK inhibitor, and a pharmaceutically acceptable excipient, A pharmaceutical composition intended for use in the treatment of BRAF V600 mutation-positive cancer in patients.

15. A kit for use according to claim 13 or a pharmaceutical composition for use according to claim 14, wherein the CSE inhibitor is defined in claim 9 and / or the MAPK inhibitor is defined in claims 6 to 8.

16. The cancer, preferably tumor, is selected from the group consisting of melanoma, skin cancer, epithelial cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, thyroid cancer, breast cancer, ovarian cancer, brain cancer, pancreatic cancer, gastrointestinal neuroendocrine tumor, neuroblastoma, glioma, astrocytoma, leukemia, hairy cell leukemia, hepatobiliary cancer, nephroblastoma (Wilms' tumor), histiocytosis, Langerhans cell histiocytosis, and Erdheim-Chester disease, preferably melanoma, colorectal cancer, colon cancer, rectal cancer, lung cancer; in particular melanoma, a CSE inhibitor for use according to claim 1 or 2, the kit according to claim 13, or the pharmaceutical composition according to claim 14.

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