Real-time tracking of apoptosis

Abstract

Compositions, kits, and methods are provided that can predict the cytotoxic or cytoprotective effect of therapeutic agents without altering the permeabilization of test cells.

Description

Technical Field

[0001] The present invention relates to prediction of the effectiveness of anticancer therapy. More specifically, the present invention provides an anti-apoptosis inhibitor combination that can be used to predict the sensitivity of a subject to anti-cancer therapy, as well as a kit and a device containing the anti-apoptosis inhibitor combination. Background Art

[0002] As more targeted therapies are approved for different types of cancer, there is a growing need for predictive biomarkers so that these therapies can be targeted to patients who will benefit most from them. Unfortunately, there are currently insufficient biomarkers available for cancer therapy.

[0003] Recently, the development of tyrosine kinase inhibitors (TKIs) has improved the treatment of patients with advanced disease. For example, EGFR mutation testing has been successfully used as a biomarker for initial treatment with EGFR inhibitors. However, many targeted drugs lack genetic predictive markers. In addition, resistance to these drugs often develops, and because the mechanisms of resistance are diverse, it is often unclear which treatment approach is best after resistance develops.

[0004] With the advent of precision medicine in cancer, new predictive biomarkers are urgently needed to assess the effectiveness of therapy for patients. In addition to standard molecular analyses, functional assays are currently being developed to guide clinical decision making.

[0005] In this regard, the use of so-called dynamic BH3 profiling or DBP for direct assessment of therapeutics has been reported.

[0006] The method is based on the use of synthetic BH3 peptides to catalyze the apoptotic cell death process. However, the actual method reported in the method requires permeabilization of cells with digitonin to allow the BH3 peptide to internalize and exert its pro-apoptotic activity. This represents a significant limitation of the assay, as it forces the cells to be fixed (and therefore killed) after incubation with the peptide, so that only one time point can be visualized. For example, see US20220163510 or US10393733.

[0007] Therefore, despite efforts made, further tools are still needed to appropriately predict responses to anticancer agents. Summary of the invention

[0008] The inventors have identified a combination of anti-apoptotic inhibitors that is capable of providing effective and sensitive predictive information regarding a subject's response to one or more specific therapies.

[0009] Specifically, the inventors have designed a combination of molecules that have an inhibitory effect on BCL-2, BCL-xL and MCL-1. As shown below, the inhibitors that make up the combination of the present invention can effectively penetrate cancer cells without damaging them. Therefore, there is no need to use a permeabilizing agent.

[0010] In addition, the inventors have found that the cell apoptosis state induced by the inhibitor combination of the present invention is at least equal to or even higher than the cell apoptosis effect provided by the DBP regimen provided in the prior art.

[0011] The present invention thus provides a combination which is at least as effective as the BH3 peptide / digitonin reported in prior art DBP protocols in determining the initiation of apoptosis and has the significant advantage that the cell viability of a sample of the test cell population is not negatively affected since in the present case no cell permeabilization is required.

[0012] The above is a huge improvement over the DBP methodology disclosed in the prior art to date.

[0013] On the one hand, the existing technology requires two steps to achieve the initiation effect of cell apoptosis: (1) increasing the permeability of the cell membrane by administering digitonin, and (2) contacting the cells with synthetic BH3 peptides to catalyze cell apoptosis.

[0014] Advantageously, by means of the combination of the present invention, a permeabilization step will be avoided, allowing continuous monitoring of living cells.

[0015] On the other hand, one of the major concerns in cancer management is not only predicting the response when a subject is about to start anticancer therapy, but also predicting how the subject will respond to long-term anticancer therapy (eg, develop resistance to therapy).

[0016] An important advantage of using the inhibitor combination of the present invention (also referred to as an "apoptosis catalyst combination") is that when the combination is administered, it can initiate a high degree of apoptotic state without negatively affecting the integrity of the cancer cells. This allows real-time monitoring of the evolution of cancer cells during long-term therapy using a single test sample. This information is very useful for appropriate management of cancer diseases at different stages, predicting the onset of potential resistance to treatment, and testing several anti-cancer therapies over time.

[0017] In summary, the combinations provided by the present invention represent a significant advance in the effective administration of anti-cancer therapy.

[0018] Thus, in a first aspect, the present invention provides a combination comprising a BCL-2 inhibitor, a BCL-xL inhibitor and a MCL-1 inhibitor.

[0019] The second aspect of the present invention provides a kit comprising the inhibitor combination defined in the first aspect of the present invention.

[0020] The inventors have developed a method that can predict a subject's sensitivity to a particular drug candidate based on one or more anti-apoptotic BCL-2 family inhibitors with a molecular weight below 2.0 KDa.

[0021] It is worth noting that in the context of the present invention, inhibitors are used as reagents rather than therapeutic drugs.

[0022] The method of the present invention is based on determining the proximity of cells to a programmed cell death (i.e., apoptosis) threshold, also referred to as determining the degree of "activation" of cancer cells to death. The method of the present invention allows identification of drugs that bring cells closer to the programmed cell death threshold (maximum apoptosis initiation). These methods of the present invention can be applied to a single clinical cancer sample so that it is easy to identify drugs that bring cells in the sample closest to the programmed cell death threshold of the single sample. The drug so identified is most likely to provide clinical benefits to the object from which the sample is derived. Therefore, the present invention provides a method for personalized treatment of a single cancer patient based on the use of one or more BCL-2 anti-apoptosis family small molecule inhibitors.

[0023] Therefore, in a third aspect, the present invention provides a method for predicting the cytotoxicity of a therapeutic agent to a cell, the method comprising: (a) contacting an isolated test cell population with a test therapeutic agent; (b) contacting the cell population obtained in step (a) with a specific concentration of one or more anti-apoptotic BCL-2 family inhibitors, wherein the molecular weight of the one or more anti-apoptotic BCL-2 family inhibitors is less than 2KDa, in particular less than 1.5KDa, and in particular less than 1KDa; (c) determining an apoptosis initiation value; (d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with the one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and (e) comparing the two initiation values; wherein: step (d) is performed in the absence of a therapeutic agent; and the process is performed in the absence of a cell permeabilizing agent; and wherein a decrease in the initiation value of the test cell portion compared to the initiation value of the reference cell portion indicates that the therapeutic agent is cytotoxic to the cells. After exposure to a BCL-2 family inhibitor, the initiation of apoptosis in the treated group is altered compared to the untreated group, resulting in an altered initiation of apoptosis in the treated cells (generally defined as Δ initiation %), which indicates that the treatment has the ability to engage apoptosis and eliminate target cells. Since the present invention allows this measurement to be performed on living cancer cells, several treatments can be administered and monitored over time as a real-time track of the engagement of apoptosis.

[0024] In a fourth aspect, the present invention provides a method for predicting the protective effect of a therapeutic agent on cells, the method comprising: (a) contacting an isolated test cell population with a test therapeutic agent; (b) contacting the cell population obtained in step (a) with a specific concentration of one or more anti-apoptotic BCL-2 family inhibitors, wherein the molecular weight of the one or more anti-apoptotic BCL-2 family inhibitors is less than 2KDa, in particular less than 1.5KDa, and in particular less than 1KDa; (c) determining a cell apoptosis initiation value; (d) obtaining a reference cell apoptosis initiation value by contacting another isolated cell population sample from the subject with one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and (e) comparing the two initiation values; wherein: step (d) is performed in the absence of a therapeutic agent; and the process is performed in the absence of a cell permeabilizing agent; and wherein, an increase in the initiation value of the test cell portion compared to the initiation value of the reference cell portion indicates that the therapeutic agent has a protective effect on the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .A) The percentage of cytochrome c retained after 16 hours of treatment with anticancer drugs and exposure to different concentrations of BIM peptide (X-axis, comparison purpose, standard DBP) in NALM-6 cell line and B) SEM cell line. C) The percentage of cytochrome c retained measured by flow cytometry (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of combination A of the present invention as a cell apoptosis catalyst (X-axis) in NALM-6 cell line and D) SEM cell line. E) Comparison between normal DBP and delta start % measured by flow cytometry RTA and microscopy RTA using a cell apoptosis catalyst in NALM-6 cell line and F) SEM cell line.

[0026] Squares = reference; circles = trametinib 100 nM in (A) and (C), or sunitinib 1000 nM in (B) and (D); triangles = imatinib 1000 nM.

[0027] In (E) and (F), black bars represent ΔActiv% measured using DBP and white bars represent ΔActiv% measured using flow cytometry RTA.

[0028] Figure 2.A) The percentage of cytochrome c retained after 16 hours of treatment with anticancer drugs and exposure to different concentrations of BIM peptide (X-axis, comparison purpose, standard DBP) in GIST-T1 cell line and B) GIST-T1 / 670 cell line. C) The percentage of cytochrome c retained measured by flow cytometry (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of combination A of the present invention as a cell apoptosis catalyst (X-axis) in GIST-T1 cell line and D) GIST-T1 / 670 cell line. E) The percentage of TMRE-positive cells measured by fluorescence microscopy (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of combination A of the present invention as a cell apoptosis catalyst (X-axis) in GIST-T1 cell line and F) GIST-T1 / 670 cell line. G) Comparison between normal DBP and Δinitiation % measured by flow cytometry RTA and microscopy RTA using apoptosis catalyst in GIST-T1 cell line and H) GIST-T1 / 670 cell line.

[0029] Squares = reference; circles = imatinib 1000 nM; triangles = gefitinib 1000 nM.

[0030] In (E) and (F), black bars represent ΔActiv% measured using DBP, grey bars represent ΔActiv% measured using flow cytometry RTA, and white bars represent ΔActiv% measured using microscopy RTA.

[0031] Figure 3 .A) The percentage of cytochrome c retained measured by flow cytometry (Y axis) after treatment with anticancer drugs for 16 hours and exposure to different concentrations of combination B of the present invention as a catalyst for apoptosis (X axis) in NALM-6 cell line and B) SEM cell line. C) The percentage of cytochrome c retained measured by flow cytometry (Y axis) after treatment with anticancer drugs for 16 hours and exposure to different concentrations of combination C of the present invention as a catalyst for apoptosis (X axis) in NALM-6 cell line and D) SEM cell line. E) Normal DBP compared with treatment with combination A (from Figure 1 ), comparison between the Δ onset % measured by flow cytometry RTA of combination B and combination C.

[0032] Squares = reference; circles = trametinib 100 nM in (A) and (C), or sunitinib 1000 nM in (B) and (D); triangles = imatinib 1000 nM.

[0033] In (E) and (F), black bars represent Δactivation % measured using DBP, dark grey bars represent Δactivation % measured using combination A at RTA, light grey bars represent Δactivation % measured using combination B at RTA, and white bars represent Δactivation % measured using combination C at RTA.

[0034] Figure 4 .A) The percentage of cytochrome c retained measured by flow cytometry after treatment with anticancer drugs for 16 hours and exposure to different concentrations of combination B of the present invention as a cell apoptosis catalyst (X axis) in GIST-T1 cell line and B) GIST-T1 / 670 cell line (Y axis). C) The percentage of cytochrome c retained measured by flow cytometry after treatment with anticancer drugs for 16 hours and exposure to different concentrations of combination C of the present invention as a cell apoptosis catalyst (X axis) in GIST-T1 and D) GIST-T1 / 670 cell lines (Y axis). E) Normal DBP compared with treatment with combination A (from) in GIST-T1 cell line and F) GIST-T1 / 670 cell line. Figure 1 ), comparison between the Δ onset % measured by flow cytometry RTA of combination B and combination C.

[0035] Squares = reference; circles = imatinib 1000 nM; triangles = gefitinib 1000 nM.

[0036] In (E) and (F), black bars represent Δactivation % measured using DBP, dark grey bars represent Δactivation % measured using combination A at RTA, light grey bars represent Δactivation % measured using combination B at RTA, and white bars represent Δactivation % measured using combination C at RTA.

[0037] Figure 5 .A) The percentage of TMRE-positive cells measured using a microfluidic platform and fluorescence microscopy (Y axis) after treatment with anticancer drugs for 16 hours in GIST-T1 cell lines and exposure to different concentrations of BIM peptides (X axis). B) The percentage of retained cytochrome c measured using flow cytometry (Y axis) after treatment with anticancer drugs for 16 hours in GIST-T1 cell lines and exposure to different concentrations of combination A of the present invention as a cell apoptosis catalyst (X axis). C) The percentage of TMRE-positive cells measured using a microfluidic platform and fluorescence microscopy (Y axis) after treatment with anticancer drugs for 16 hours in GIST-T1 cell lines and exposure to different concentrations of combination A of the present invention as a cell apoptosis catalyst (X axis). D) Comparison between normal DBP in a microfluidic chip, microscopy RTA using combination A in a microfluidic chip, and Δ start % measured using RTA using combination A.

[0038] Squares = reference; circles = imatinib 1000 nM; triangles = gefitinib 1000 nM.

[0039] Black bars represent ΔActuation % measured using DBP within the microfluidic chip, grey represents ΔActuation % measured using microscopy RTA within the microfluidic chip using combination A, and white represents ΔActuation % measured using RTA within the microfluidic chip using combination A. DETAILED DESCRIPTION

[0040] All terms used in this application, unless otherwise specified, are to be understood according to their common meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the specification and claims, unless otherwise explicitly specified to provide a broader definition.

[0041] For the purposes of the present invention, any given range includes the lower and upper limits of that range.

[0042] As used herein, the meaning of the term "comprising" encompasses three options, namely, "including," "consisting of," and "consisting essentially of."

[0043] The present invention provides in a first aspect a combination comprising a molecule that inhibits BCL-2, BCL-xL and MCL-1.

[0044] In the context of the present invention, the term "inhibitor" includes both molecules that have a direct effect on the target (e.g., by binding; direct inhibitors) and molecules that initiate a mechanism that provides an inhibitory or down-regulatory effect on BCL-2, BCL-xL and MCL-1 (indirect inhibitors). As shown below, whether direct inhibitors or indirect inhibitors are included, the relevant technical feature is that the combination must inhibit BCL-2, BCL-xL and MCL-1, and as described above, this simultaneous inhibition is an essential feature for inducing an apoptotic state and determining the initiation of apoptosis.

[0045] Those skilled in the art know how to determine whether a molecule is an inhibitor of any of these targets, and a number of publications have reported the identification and use of molecules that inhibit BCL-2, BCL-xL, and MCL-1.

[0046] In the context of the present invention, a combination may include one molecule that is specific for each of the targets, i.e., the combination will include 3 molecules. In other cases, a combination may include a molecule that exerts a dual effect on two of the targets (e.g., a BCL-2 / BCL-xL inhibitor). In this alternative case, the combination will include two molecules for inhibiting BCL-2, BCL-xL, and MCL-1: one dual-acting and the other specific for the other target.

[0047] The intrinsic pathway, also known as the mitochondrial pathway, is regulated by the BCL-2 family of proteins. This family has different members that can be classified based on structure, function, and the BCL-2 homology (BH) domain: activator members, antiapoptotic members, sensitizers, and effectors.

[0048] In the context of the present invention, the molecules incorporated into the combinations are those that bind with high affinity and specificity to the hydrophobic groove of the anti-apoptotic protein targets, thereby inhibiting them.

[0049] There are currently known inhibitors for each of the three families (BCL-2, BCL-xL and MCL-1). Some of these inhibitors will be explained in detail below by way of example (see also Table 1).

[0050] Molecules that directly inhibit BCL-2

[0051] The first molecule directed against BCL-2 was HA14-1, which showed activity in vitro and in vivo when used alone or in combination with cytotoxic therapy.

[0052] Abbott Laboratories (now AbbVie) developed ABT-737, the first specific BH3 analog to hit the target, blocking the BH3 binding domain and inhibiting BCL-2, BCL-xL, and BCL-W. The compound was further refined to improve oral bioavailability, resulting in the analog ABT-263 (navitoclax).

[0053] Navitoclax has shown promising results in a variety of blood cancers, particularly chronic lymphocytic leukemia (CLL), which expresses high levels of BCL-2.

[0054] To reduce the risk of thrombocytopenia, AbbVie has developed a selective BCL-2 inhibitor called ABT-199 (venetoclax). Clinical responses to venetoclax in CLL patients have shown that venetoclax has impressive anticancer activity, leading to rapid tumor reduction and even tumor lysis syndrome.

[0055] Other illustrative non-limiting examples of BCL-2 inhibitors are APG-1252, BGB-11417, S55746, or SPC2996, among others.

[0056] The examples provided below are based on combinations comprising one of ABT-199 or S55746 as BCL-2 inhibitors. The same results provided by ABT-199 or S55746 (chemically unrelated) can also be obtained using any of the above inhibitors from the same family, since they act on the same target and are very small in size so that they can also diffuse across the cell membrane.

[0057] Molecules that directly inhibit BCL-xL

[0058] Selective BCL-xL inhibitors are also being developed for their potential anticancer activity. One of the first compounds that proved highly selective was WEHI-539, which was also effective against solid tumors. More recent derivatives of this first BCL-xL inhibitor have also been reported, such as A-1155463 or A-1331852.

[0059] Newer derivatives of this first BCL-xL inhibitor, such as A-1155463 or A-1331852, have also been investigated with promising preclinical results.

[0060] The latter, in particular, holds great promise due to its oral bioavailability. In contrast, a novel strategy to target BCL-xL degradation using a proteolysis targeting chimera (PROTAC; DT2216) is currently being evaluated, which has unique selectivity for targeting anti-apoptotic proteins in tumor cells but not in platelets, and has clinical potential and will also be part of the scope of this invention.

[0061] The embodiments provided below are based on a combination comprising one of A-1331852 or WEHI-539 as a BCL-xL inhibitor. The same results as provided by the inclusion of A-1331852 or WEHI-539 can also be obtained using any of the above inhibitors from the same family, since they act on the same target and are very small in size so that they can also diffuse across the cell membrane effortlessly.

[0062] Molecules that directly inhibit MCL-1

[0063] The observation that the MCL-1 antiapoptotic protein is often used by cancer cells to evade apoptosis has also stimulated the development of novel targeted therapies.

[0064] One of the first selective inhibitors was A-1210477, which showed excellent in vitro effects in hematological malignancies and solid tumors (such as breast cancer and lung cancer cell lines), especially when used in combination with navitoclax. Inspired by the anticancer effects of these molecules, a new generation of promising small molecule MCL-1 antagonists is currently in clinical development. These include S64315 / MIK665, AZD-5991, PRT1419 and AMG-176, which are currently being explored in clinical trials (NCT02992483, NCT04629443, NCT03013998, NCT02675452, NCT04178902, NCT04543305, etc.), mainly for the treatment of hematological malignancies. Other compounds that inhibit or degrade MCL-1 have also shown activity in preclinical models. Given that MCL-1 is a short-lived protein, indirect targeting or protein-selective degradation approaches using CDK9 inhibitors such as avocidib, AZD4573 (NCT03263637), or voruciclib (NCT03547115) have emerged as alternative strategies. BFL-1 / A1 can similarly be targeted using CDK9 inhibitors or dual inhibitors of MCL-1 and BFL-1.

[0065] S63845 has also been reported to act as a highly selective MCL-1 inhibitor with no cross-reactivity to BCL-2 or BCL-xL.

[0066] The examples provided below are based on combinations comprising either S63845 or AZD5991 as MCL-1 inhibitors. Similar results can be obtained using any of the above small molecule inhibitors as they can also diffuse across the cell membrane.

[0067] Indirect targeting of antiapoptotic BCL-2 proteins

[0068] Antiapoptotic proteins can also be disrupted using molecules that are unable to directly inhibit the BH3-binding domains of BCL-2, BCL-xL, and MCL-1. In this regard, different strategies have been developed to target these proteins.

[0069] For example, small molecules that directly activate the effector BAX (including BTSA1, BAM7, or BAI1) will indirectly block all anti-apoptotic proteins.

[0070] The use of antisense oligonucleotides (such as BP1002, G3139 or oblimersen sodium) will block or alter the mRNA encoding anti-apoptotic proteins and impair their synthesis.

[0071] PNT2258 is a 24-mer DNA oligodeoxynucleotide specific for the BCL-2 promoter, which can lead to downregulation of the mRNA encoding BCL-2, thereby reducing BCL-2 levels.

[0072] Different drugs that inhibit other proteins may indirectly cause changes in specific pathways, leading to altered expression of anti-apoptotic proteins, including but not limited to reduction of MCL-1 by inhibition of CDK9 by dinaciclib, AZD4573, or avosidide, or reduction of BCL-2 expression by the steroid antiprogestin mifepristone.

[0073] Use a PROTAC construct (e.g., DT2216) that tags BCL-xL for degradation by the proteasome, thereby reducing the level of the protein.

[0074] Table 1

[0075]

[0076]

[0077]

[0078]

[0079] A person skilled in the art can easily select inhibitors from each of these three groups using only their common sense, or use promiscuous anti-apoptotic inhibitors (i.e., small molecules that can inhibit several anti-apoptotic proteins at the same time, such as ABT-263, APG-1252, AZD0466, etc.) and combine them to obtain similar results.

[0080] In one embodiment of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the BCL-2 inhibitor is ABT-199 or S55746.

[0081] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the BCL-xL inhibitor is a direct inhibitor. In an alternative embodiment, the BCL-xL inhibitor is an indirect inhibitor. In another embodiment, the BCL-xL inhibitor is A-1331852 or WEHI-539.

[0082] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the MCL-1 inhibitor is a direct inhibitor, such as an inhibitor that binds to the BH3 binding groove of MCL-1, in particular S63845, or AZD5991 or DT2216.

[0083] In one embodiment of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the inhibitor of BCL-2, BCL-xL and MCL-1 is a direct inhibitor.

[0084] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the inhibitor of BCL-2 and MCL-1 is a direct inhibitor, and the inhibitor of BCL-xL is an indirect inhibitor.

[0085] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises ABT-199, A-1331852 and S63845; or, alternatively comprises S55746, WEHI-539 and AZD5991; or, alternatively comprises ABT-199, DT2216 and AZD5991.

[0086] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the combination consists of ABT-199, A-1331852 and S63845; or, alternatively, consists of S55746, WEHI-539 and AZD5991; or, alternatively, consists of ABT-199, DT2216 and AZD5991.

[0087] In another embodiment of the first aspect of the invention, optionally in combination with any embodiment provided above or below, the combination includes one or more other types of anti-apoptotic BCL-2 family inhibitors, such as activators of pro-apoptotic members of the BCL-2 family (particularly BAX, BAK1, BIM, BID and BBC3 (commonly referred to as PUMA)), and inhibitors of anti-apoptotic members of the same family (e.g., BCL-2, BCL-xL, BCL-W and MCL-1). The molecular weight of these other inhibitors is less than 2KDa, more specifically less than 1.9KDa, less than 1.8KDa, less than 1.7KDa, less than 1.6KDa, less than 1.5KDa, less than 1.4KDa, less than 1.3KDa, less than 1.2KDa, less than 1.1KDa or less than 1.0KDa.

[0088] Activator proteins (BIM, BID, and PUMA) bind and directly activate the central effectors of apoptosis, BAX and / or BAK1, which subsequently undergo conformational changes and oligomerize, forming pores in the mitochondrial membrane and causing MOMP.

[0089] The function of the sensitizer proteins (BAD, PMA-induced protein 1 (commonly known as NOXA), BIK, BMF, and HRK) is to inhibit anti-apoptotic proteins and / or displace activators (e.g., BIM, or monomers of BAX or BAK1) that may ultimately lead to apoptosis. The anti-apoptotic members of the BCL-2 family have four BCL-2 homology domains that form a binding groove that sequesters activators or sensitizers from BAX and BAK1. The BH3-only proteins PUMA, NOXA, BID, and BIM have a strong affinity for and inhibit MCL-1, while BAD has a stronger affinity for BCL-2, BCL-W, and BCL-xL than for MCL-1.

[0090] In the context of the present invention, "anti-apoptotic BCL-2 family inhibitors" are understood to be any known small molecule that directly (binds to BCL-2) or indirectly has a negative effect on BCL-2 cell activity, this negative effect is because BCL-2 is eliminated / degraded, or even if BCL-2 is produced, its action is inhibited by binding. In another embodiment of the first aspect of the present invention, optionally combined with any embodiment provided above or below, the combination includes one or more other types of anti-apoptotic BCL-2 family inhibitors, rather than the inhibitors belonging to BCL-2 inhibitors, BCL-xL inhibitors and / or MCL-1 inhibitors mentioned in claim 1. The preparation of the combination is conventionally carried out, mixing small molecules under stirring. These molecules must be added at concentrations at which they provide an inhibitory effect on the target. This concentration usually corresponds to the concentration recommended by the manufacturing company. However, if necessary, a person skilled in the art can easily use only conventional tests to accurately adjust the concentration of a specific inhibitory molecule. For example, based on the Ki value.

[0091] Examples of anti-apoptotic BCL-2 family inhibitors include, but are not limited to, HA14-2, obacla, gossypol (AT-101), oblimersen, ABT-737, ABT-263 (navitoclax), ABT-199 (venetoclax), APG-1252, APG-2575, AZD0466, BGB-11417, NU-0129, S55746 / BCI -201, SPC2996, WEHI-539, A1155463, A1331852, DT2216, S-055746, A-1210477, AMG-17 6. AMG-397, AZD-5991, PRT1419, S64315 / MIK665, S63845, MIM1, VU661013, GDC-0941, etc.

[0092] The inhibitors forming part of the combination will be included in an amount sufficient to provide an inhibitory effect. The required amount of each inhibitor will be known and can be easily adjusted by those skilled in the art.

[0093] Reagent test kit

[0094] All the above embodiments involving inhibitor combinations provided in the first aspect of the present invention are also embodiments of the kit of the second aspect of the present invention.

[0095] In one embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises a vial containing a mitochondrial buffer and instructions for use.

[0096] In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the combination includes one or more other types of anti-apoptotic BCL-2 family inhibitors, such as those mentioned above, such as activators of pro-apoptotic members of the BCL-2 family (e.g., BAX, BAK1, BIM, BID, and BBC3 (commonly known as PUMA)), and inhibitors of anti-apoptotic members of the same family (e.g., BCL-2, BCL-XL, BCL-W, and MCL-1), as well as other inhibitors and activators of the BCL-2 family. Illustrative examples have been provided above.

[0097] In another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises each inhibitor in a separate compartment, and instructions for preparing the combination of the invention. Alternatively, in another embodiment of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit already comprises the inhibitor combination of the first aspect of the invention.

[0098] In another embodiment of the third aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the kit is a microfluidic device.

[0099] Microfluidic devices are easy to manufacture, low-cost, and robust, and can be used in hospitals on a long-term basis to routinely perform predictive cancer therapy response assays at very low cost. In addition, since the requirements for cells are lower than with existing technologies, these analyses can be performed in biopsy samples obtained non-surgically (such as fine needle aspirates). From a clinical perspective, this approach may represent a breakthrough in how cancer patients are treated and can greatly improve clinical outcomes, even for the most complex cases (recurrent / refractory / metastatic tumors).

[0100] Any commercially available microfluidic device may be used in the context of the present invention, as long as the manufacturer's instructions are followed.

[0101] Chemical gradients play a key role in many biological processes and regulate many cellular functions in vivo. In fact, there are multiple examples of gradient-dependent phenomena in nature, such as chemotaxis, which triggers cell migration by establishing a gradient. In addition, chemical gradients have been shown to influence various cellular behaviors, such as tumorigenesis.

[0102] Therefore, it is clear that studying and possibly reproducing these conditions in vitro is crucial for life science related research.

[0103] In one embodiment of the second aspect of the invention, the kit is a microfluidic device comprising means for generating a gradient (whether logarithmic or linear, in particular logarithmic) of the inhibitor combination of the invention. This allows for replication of dosage curves.

[0104] The two most common ways to generate chemical gradients in microfluidics are: (i) using specially (ad hoc) designed microchannels with at least two inlets, in which adjacent streams containing different concentrations of the chemical of interest (i.e., the inhibitor combination defined in the first aspect of the invention) are mixed to generate a gradient, and (ii) utilizing the ability of cells to secrete and consume biochemical substances in their vicinity, combined with slow perfusion to generate a gradient in a direction parallel to the direction of fluid flow.

[0105] Those skilled in the art can already select the most suitable device in the commercial device with gradient function.If those skilled in the art want to design their own device with gradient, then using their general knowledge, they just can select a kind of device that is more suitable for producing this gradient in different devices.In one embodiment, optionally in combination with any embodiment provided above or below, the microfluidic device comprises a gradient generator, particularly a T-junction gradient generator.

[0106] In this embodiment, the T-junction at the end of the microchannel is a convection cell. In order to decouple the convection through the side channel from the diffusion process through the main channel, the total flow through the inlet and outlet must be matched. Assuming that one convection cell carries a constant concentration of solute and the other cell carries a buffer, this allows a gradient to be generated in the microchannel, which remains constant as long as the flow and concentration of the solute in each convection cell remain constant.

[0107] The inventors validated the device in cell lines and patient tumor samples of human gastrointestinal stromal tumors (GIST). GIST is a genomic-driven tumor that is clinically treated with small molecule inhibitors targeting KIT and PDGFRA receptor tyrosine kinases. This novel technical approach allows non-invasive, rapid and convenient therapy evaluation of cells isolated from patients (even from biopsies obtained non-surgically) directly in an accurate and highly reproducible manner. In addition, since this 1 measurement is fully integrated in the microfluidic chip, these analyses can be standardized to require minimal operation, making it available as a routine test in hospitals in the future.

[0108] Furthermore, the versatility of this microchip enables monitoring of patient tumors to adjust treatment strategies throughout disease progression.

[0109] Utilizing this new approach, continued personalized cancer treatment will be achieved through implementation of functional assays in the clinic, thereby facilitating precision medicine to overcome cancer resistance to therapy and avoid relapse.

[0110] Since the equipment required to perform the assay requires only an unsophisticated microfluidic pump and a fluorescence readout device, this new technology could be easily integrated into a ready-to-use medical device for on-site analysis in hospitals.

[0111] method

[0112] In a third aspect, the invention provides a method for predicting the cytotoxicity of a therapeutic agent to a cell.

[0113] In a fourth aspect, the invention provides a method of predicting cytoprotection of a cell by a therapeutic agent.

[0114] All the embodiments provided above in the first aspect or the second aspect of the present invention are also embodiments of the methods of the third aspect and the fourth aspect of the present invention.

[0115] In a first step, the method of the invention comprises contacting an isolated test cell population with a test therapeutic agent.

[0116] The test cell population is isolated from a subject by using any conventional technique and cultured using well-known standardized culture media.

[0117] Since the requirements for cells are lower than those of existing technologies, the method of the present invention can also be performed on biopsies obtained non-surgically (such as fine needle aspirates). From a clinical perspective, this method could represent a breakthrough in how cancer patients are treated and could greatly improve clinical outcomes, even for the most complex cases.

[0118] In one embodiment of the method for the third aspect and the fourth aspect of the present invention, optionally in combination with any embodiment provided above or below, the cell is from a subject known to suffer from or suspected of suffering from cancer. The subject is preferably a mammal. Mammals are, for example, humans, non-human primates, mice, rats, dogs, cats, horses or cattle. In an alternative embodiment, optionally in combination with any embodiment provided above or below, the subject has previously been diagnosed as suffering from cancer and may have received cancer treatment. Or, the subject has not previously been diagnosed as suffering from cancer.

[0119] The test cell population is then exposed to the therapeutic agent.

[0120] In one embodiment of the method of the third aspect and the fourth aspect of the present invention, optionally in combination with any embodiment provided above or below, the therapeutic agent induces apoptosis. In one embodiment of the method of the third aspect and the fourth aspect of the present invention, optionally in combination with any embodiment provided above or below, the therapeutic agent is a chemotherapeutic agent. In another embodiment of the method of the third aspect and the fourth aspect of the present invention, optionally in combination with any embodiment provided above or below, the therapeutic agent is a targeted chemotherapeutic agent. In another embodiment of the method of the third aspect and the fourth aspect of the present invention, optionally in combination with any embodiment provided above or below, the targeted chemotherapeutic agent is a kinase inhibitor, such as an inhibitor of MEK1 and MEK2 kinase activity, an EGFR tyrosine kinase inhibitor, or an inhibitor of kinase (such as BCR-ABL, ABL and KIT). In another embodiment of the method, other therapeutic agents are chemotherapeutic agents, such as doxorubicin, cisplatin, 5-FU and other cytotoxic agents used as anticancer agents. In another embodiment of the method, other therapeutic agents are antibodies, including immune checkpoint inhibitors, such as pembrolizumab, rituximab, trastuzumab, etc. In another embodiment of this method, the other therapeutic agent is an engineered immune cell, such as a chimeric antigen receptor CAR-T cell therapy.

[0121] Next, the test cell population that has been exposed to the test therapeutic agent is contacted with one or more BCL-2 family apoptosis inhibitors mentioned and defined in the previous embodiments. This step is performed using conventional means in the prior art, using experimental conditions that are well established or can be easily optimized by those skilled in the art. An illustrative method is provided below in the Examples section, where cells are contacted with the combination at 37°C for an appropriate period of time to ensure that apoptosis is induced.

[0122] As already explained above, in the context of the present invention, the expression "anti-apoptotic BCL-2 family inhibitor" encompasses any small molecule (molecular weight less than 1.9 KDa, less than 1.8 KDa, less than 1.7 KDa, less than 1.6 KDa, less than 1.5 KDa, less than 1.4 KDa, less than 1.3 KDa, less than 1.2 KDa, less than 1.1 KDa or less than 1.0 KDa) that directly or indirectly inhibits BCL-2 function or induces BCL-2 elimination. Among potential targets, the pro-apoptotic members BAX, BAK1, BIM, BID and BBC3 (commonly known as PUMA) can be found, as well as anti-apoptotic members of the same family such as BCL-2, BCL-xL, BCL-W, BFL-1 and MCL-1. BH3-only proteins are a subclass of pro-apoptotic BCL-2 proteins that contain only one BCL-2 homology domain and can be divided into activators and sensitizers. Activator proteins (BIM, BID, and PUMA) bind and directly activate BAX and / or BAK1, the central effectors of apoptosis, which then undergo conformational changes and oligomerize, forming pores in the mitochondrial membrane and causing MOMP. The function of sensitizer proteins (BAD, PMA-induced protein 1 (commonly known as NOXA), BIK, BMF, and HRK) is to inhibit anti-apoptotic proteins and / or displace activators (such as BIM, or monomers of BAX or BAK1) that may ultimately lead to apoptosis. Anti-apoptotic members of the BCL-2 family have four BCL-2 homology domains that form a binding groove that sequesters activators or sensitizers from BAX and BAK1. The BH3-only proteins PUMA, NOXA, BID, and BIM have a strong affinity for and can inhibit MCL-1, while BAD has a stronger affinity for BCL-2, BCL-W, and BCL-xL than for MCL-1.

[0123] There are some well-known molecules that can modulate the above targets. In addition to those mentioned above, they also include HA14-2, obakla, gossypol (AT-101), obimersen, ABT-737, ABT-263 (navitoclax), ABT-199 (venetoclax), APG-1252, APG-2575, AZD0466, BGB-11417, NU-0129, S55746 / BCI-201, SPC2996, WEHI-539, A1155463, A1331852, DT2216, S-055746, A-1210477, AMG-176, AMG-397, AZD-5991, PRT1419, S64315 / MIK665, S63845, MIM1, VU661013, GDC-0941, etc.

[0124] In one embodiment of the method of the third aspect and the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the triggering value is determined at different increasing concentrations of one or more anti-apoptotic BCL-2 family inhibitors to obtain a triggering curve (dose-response) for the test sample and the reference sample. In this embodiment:

[0125] - when the start-up curve of the test sample is shifted to the left relative to the start-up curve of the reference sample, this indicates that the test therapeutic agent is cytotoxic to the cells; and

[0126] - When the activation curve for the test sample is shifted to the right relative to the activation curve for the reference sample, this indicates that the test therapeutic agent protects the cells.

[0127] In one embodiment of step (b), optionally in combination with any of the embodiments provided above or below, the population of cells is contacted with a combination as defined in the first aspect of the invention or in its embodiments.

[0128] Step (c) of the method of the third aspect or the fourth aspect of the present invention determines apoptotic cell death in the sample. Many well-established protocols known to those skilled in the art can determine the extent of apoptosis in cells (see, Banfalvi G. "Methods to detect apoptotic cell death", Apoptosis, 2017 Feb; 22 (2): 306-323), most of which are based on detecting abnormalities in cell membranes (e.g., plasma membranes or mitochondrial membranes).

[0129] In one embodiment of the third aspect of the invention, optionally in combination with any embodiment provided above or below, step (c) comprises or consists of measuring the mitochondrial outer membrane permeability of the test cell population.

[0130] Outer membrane permeability can be measured by a variety of methods, such as by the loss of mitochondrial membrane potential. For example, the loss of mitochondrial membrane potential is measured by measuring molecules released from the mitochondrial intermembrane space. Examples of molecules that can be measured include cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2, or apoptosis inducing factor (AIF).

[0131] The release of molecules from the mitochondrial intermembrane space can be measured by methods known in the art. For example, by using antibodies to the molecules to be measured, i.e., antibodies to cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2, or apoptosis inducing factor (AIF). Detection can be performed by, for example, ELISA, FACS, immunoblotting, immunofluorescence, or immunohistochemistry.

[0132] In one embodiment of the third aspect of the invention, optionally in combination with any embodiment provided above or below, step (c) comprises, or consists of, determining the percentage of cytochrome C released from mitochondria.

[0133] In addition to measuring molecules released from the mitochondrial intermembrane space, other intracellular and extracellular markers can also be measured. This enables the differentiation of cell subpopulations.

[0134] Alternatively, cells may be treated with a potentiometric dye or a radioactive dye to determine outer membrane permeabilization prior to step (c).

[0135] Advantageously, a titration curve can be generated by comparing treated cells to reference cells to assess the ability of a therapy to induce apoptosis.

[0136] In view of the above, in one embodiment of the third aspect of the present invention, optionally in combination with any embodiment provided above or below, before step (c), the cell population is first contacted with a potentiometric dye or a radioactive dye. An illustrative non-limiting example of a potentiometric dye is hydrogen rhodamine 123, or tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE); in particular tetramethylrhodamine (TMRE).

[0137] JC-1 is a lipophilic cationic dye that enters mitochondria at a rate proportional to the mitochondrial inner membrane potential. JC-1 exists as a monomer at low membrane concentrations. However, JC-1 accumulates in the mitochondrial matrix under conditions of higher mitochondrial potentials. At these higher concentrations, JC-1 forms red fluorescent "J aggregates". As a monomer, the absorption / emission maximum of the dye is 527nm, while at high membrane potentials, the emission maximum is 590nm. Therefore, the ratiometric measurement of this cyanine dye emission can be used as a sensitive measurement of mitochondrial membrane potential. The dye enables dual measurement of dye concentration without measuring nuclear or cytoplasmic reference values. Studies using isolated mitochondria have shown that the 527nm emission of monomeric JC-1 increases almost linearly with membrane (M) potentials in the range of 46mV to 182mV, while the emission of the 590nm J aggregates is less sensitive to M values ​​less than 140mV and is very sensitive to potential values ​​in the range of 140mV to 182mV (Di Lisa et al., 1995). Optical filters designed for fluorescein and tetramethylrhodamine ethyl ester (TMRE) can be used to visualize the monomeric and J-aggregate forms separately. Alternatively, both forms can be viewed simultaneously using a standard fluorescein longpass optical filter set.

[0138] Dihydrorhodamine 123 is an uncharged, non-fluorescent agent that can be converted into the fluorescent laser dye rhodamine 123 (R123) by oxidation.

[0139] The measurement of the amount of mitochondrial outer membrane permeability of the test population and the reference population can be performed simultaneously. There is no mandatory sequential order for these steps.

[0140] Alternatively, step (c) comprises or consists of measuring the activation of caspases.

[0141] Caspases (cysteine-aspartic acid proteases) are involved in the early stages of apoptosis. Activation of caspases is a key event in the early apoptotic process. The molecular events leading to caspase activation have been used in cell-free systems to detect apoptosis produced in the mitochondrial death pathway. Caspase activation can be detected by in vitro enzyme assays. Western blot mixtures have been designed to detect proteins synthesized in response to apoptotic stimuli, such as caspases.

[0142] When the starting material is RNA, quantitative reverse transcription PCR (RT-qPCR) is used. For RT-qPCR analysis of gene expression, activated (cleaved) effector caspases 3 and 7, activated initiator caspases 2, 8 and 9, APAF, BAX, BAK, BID, PARP are recommended. In addition to early detection of apoptosis by Western blot analysis, caspase activity can be detected by cleavage of caspase substrates in vivo, flow cytometry, fluorescence and light microscopy, and by fluorescent ELISA. Fluorescent substrates or fluorescent antibodies that preferentially target caspase 3 and caspase 7 produce fluorescence that is proportional to caspase activation. Caspase-cleaved cytokeratin 18 (M30) is a specific antibody for the early stages of apoptosis that does not react with intact or necrotic cells. Detection of M30 is a reliable indicator of epithelial cell apoptosis. Another antibody, anti-PARP, can be used to detect PARP fragments and intact PARP in extracts of apoptotic cells and in extracts of healthy cells in Western blots. Cleavage of PARP by caspases can be measured by Western blotting, immunohistology, and immunoprecipitation. p53, Annexin V, and M30 proteins are detected in a similar manner.

[0143] Alternatively, step (c) may be performed by measuring extracellular phosphatidylserine.

[0144] Annexin A5 or Annexin V is a member of calcium-dependent phospholipid binding proteins that specifically adheres to phosphatidylserine (PS). In healthy cells, PS is only located on the cytoplasmic side of the plasma membrane. In the early stages of apoptosis, the asymmetric distribution of PS disappears and PS is transferred to the extracellular leaflet of the membrane. Fluorescently labeled Annexin V can be used to detect the presence of PS on the extracellular side of the cell membrane. Suitable dyes are membrane-impermeable dyes, such as propidium iodide (PI), trypan blue, 7-aminoactinomycin (7-AAD).

[0145] The amount of mitochondrial apoptotic death obtained from the test cell population is compared to the amount of mitochondrial apoptotic death obtained from a reference cell population that has not been contacted with the therapeutic agent.

[0146] Any statistical method may also be used to manage the measurements obtained.

[0147] The reference cell population can be a test cell population isolated from the same subject but not contacted with the therapeutic agent. Alternatively, the reference cell population is a cell population of the same nature as the test cell population but from a healthy subject. The expression "same nature" of the present invention means that the sample is isolated from the same organ / sample of the same nature.

[0148] In one embodiment of the method of the third aspect of the invention, the kit as defined in the second aspect of the invention is used alone or in combination with any of the embodiments provided above.

[0149] In another embodiment, the method of the third aspect of the invention comprises using a microfluidic device as defined above. In this embodiment, step (a) can be performed inside or outside the microfluidic device. In one embodiment, step (a) is performed outside the microfluidic device.

[0150] According to the method, step (b) is performed in a microfluidic device and includes filling the wells with the resulting cells and perfusing the wells with a buffer containing one or more BCL-2 anti-apoptotic family inhibitors. In one embodiment, optionally in combination with any of the embodiments provided above or below, the perfusion of the BCL-2 inhibitor is performed by generating a concentration gradient. In another embodiment, optionally in combination with any of the embodiments provided above or below, a concentration gradient is formed by perfusing the culture medium only through one inlet of the device and perfusing the culture medium and one or more BCL-2 inhibitors through a second inlet of the device.

[0151] Steps (c) to (e) are performed as explained in detail above.

[0152] In another embodiment of the method of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell population is a cancer cell population and the therapeutic agent is a chemotherapeutic agent, as explained in detail above.

[0153] Example

[0154] Cell culture and treatment

[0155] GIST-T1 and GIST-T1 / 670 were kindly provided by Dr. César Serrano of the Valle de Efron Institute of Oncology. These cells were cultured at 37°C and 5% CO2 in IMDM medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 15% fetal bovine serum (FBS) (10270106, Thermo Fisher Scientific), 1% penicillin / streptomycin (15140122, Gibco, Thermo Fisher Scientific) and 1% L-glutamine (25030024, Thermo Fisher Scientific).

[0156] NALM-6 and SEM cell lines were kindly provided by the laboratory of Professor Pablo Menéndez at the Josep Carreras Leukaemia Institute. Both cell lines were cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) containing 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% L-glutamine (25030, Thermo Fisher, Gibco) and 1% penicillin / streptomycin (15140, Thermo Fisher, Gibco). Cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0157] Imatinib and gefitinib were purchased from LC Laboratories (Woburn, MA, USA); trametinib was purchased from SelleckChem (Munich, Germany); ABT-199, A-1331852, and S6385 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

[0158] A 1 mM BH3 mimetic combination was prepared by adding 0.1 mM ABT-199, 0.1 mM A-1331852, and 1 mM S63845.

[0159] Dynamic BH3 profiling using flow cytometry (for comparative purposes)

[0160] BCP-ALL and GIST cells were pretreated with different anticancer drugs for 16 h, stained with Zombie Violet to mark live cells, washed with phosphate-buffered saline (PBS), and divided into 9 different test tubes, resuspended in mannitol assay buffer (MEB) (150 nM mannitol, 10 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, and 5 mM succinate) containing 0.001% digitonin and 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM of BIM peptide, 25 μM alamethicone, and DMSO alone as a control. After incubation for 1 h at room temperature, cells were fixed with 8% formaldehyde, neutralized with N2 buffer (1.7 M tris base, 1.25 M glycine, pH 9.1), and incubated with 1:1000 of a 50 μL lysate containing AlexaFluor The cells were stained with intracellular staining buffer (PBS solution containing 1% Tween20 and 5% BSA) conjugated to cytochrome c antibody. After incubation at 4°C overnight, the results were acquired using an LSRII flow cytometer and processed using FlowJo software according to the manufacturer's instructions. A gate was set for the % of cytochrome c retained in the untreated condition and all conditions applied.

[0161] Real-time tracking of apoptosis (RTA) using flow cytometry (present invention)

[0162] Cells were seeded in 12-well plates and treated with different anticancer drugs for 16 hours. After incubation, cells were digested with trypsin, stained with Zombie Violet and washed with PBS. Afterwards, cells were resuspended in appropriate complete medium, and 25 μL of each cell solution was added to a 96-well plate containing 25 μL of complete medium solution and different apoptosis catalysts of different dilutions (including 1X ABT-199, 1X A1331852 and 10X S63845 combination A; 1X S55746, 1X WEHI-539 and 10X AZD5991 combination B; and 1X ABT-199, 1X DT2216 and 10X AZD5991 combination C). In all combinations, the concentrations used were 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, and DMSO alone. Plates for the BCP-ALL cell line were incubated at 37°C for 1 hour, plates for the GIST cell line were incubated at 37°C for 3 hours, and stained according to the following protocol: fixation with 8% PFA, neutralization with N2 buffer, and staining with antibodies against cytochrome c. Since BCP-ALL are non-adherent cells, results were obtained using an LSRII flow cytometer after overnight incubation at 4°C and processed using FlowJo software. A gate was set for the % of cytochrome c retained in the untreated condition and in all conditions applied. The same protocol but replacing the complete medium with medium without FBS was also performed.

[0163] RTA using fluorescence microscopy (present invention)

[0164] For each treatment condition, 100,000 cells were resuspended in 1 mL of complete medium, treated with the corresponding anticancer drug, and 100 μL of the cell solution was inoculated in 8 wells of a 96-well plate. The cells were incubated for 16 hours. After incubation, the cells were stained with complete medium containing 2 μM calcein AM and 100 nM TMRE (the medium used in cell culture) at 37°C for 30 minutes, washed with PBS, and exposed to the conditions of the apoptosis catalyst combination of the present invention that were the same as those using flow cytometry. The culture dish was incubated at 37°C for 3 hours, and images were taken with a Nikon Eclipse Ti microscope. Using CellProfiler, cells were automatically identified by the calcein AM field, and the TMRE intensity was quantified using an intensity threshold of the 90% percentile of the fluorescence intensity under the control condition to distinguish between apoptotic cells and non-apoptotic cells.

[0165] SU8 mold manufacturing

[0166] To develop the mold, a silicon wafer (4” n-type <100> , MicroChemicals GmbH, Ulm, Germany) was placed in a PCD-002-CE plasma cleaner (Harrick Plasma, Ithaca, NY, USA) for 20 minutes at 6.8 W power and then placed in a hot plate for 5 minutes at 95°C. Next, SU-8 photoresist (2100, MicroChem Lab, Westborough, MA, USA) was spin-coated on the wafer (first spinning at 500 rpm for 5 seconds with an acceleration of 100 rpm / s, then spinning at 3000 rpm for 30 seconds with an acceleration of 401 rpm / s) to obtain a 100 μm thick layer. The wafer was soft-baked at 65°C for 5 minutes and then at 95°C for 20 minutes to evaporate the solvent. Using a negative photoresist mask printed on a high-quality acetate film, 240 mJ / cm 2 The patterns with microfluidic chip designs were obtained by irradiation with energy of . The wafers were exposed to 65°C on a hot plate for 5 minutes and then to 95°C for 10 minutes to crosslink the photoresist. Then, they were immersed in SU-8 developer (Y020100, MicroChem Lab) for 10 minutes and cleaned with 2-propanol to remove unstable photoresist. Finally, the wafers were placed on a hot plate at 150°C for 60 minutes and finally the temperature was reduced until it reached room temperature, at which point they were silanized to obtain a hydrophobic surface.

[0167] Fabrication of microfluidic chips

[0168] To obtain the microfluidic chip, PDMS was prepared by mixing Sylgard 184 prepolymer (Dow Corning, Midland, MI, USA) with a curing agent in a ratio of 1:10 and then degassing in a cavity for 1 h. Three different layers were prepared by calculating the volume of the polymer to obtain the desired PDMS layer thickness. For the first layer of 2 mm thickness, the PDMS prepolymer was poured onto a SU8 master mold with a designed pattern fixed inside a plastic Petri dish; for the second layer of 1 mm thickness, the prepolymer was dispensed directly into an empty Petri dish; for the third layer, a clean 75x50 mm glass slide (CLS294775X50, Sigma-Aldrich) was pressed onto the uncured polymer mixture to obtain a thin PDMS layer on the glass. The PDMS was placed on a flat surface at room temperature and then kept at 85 °C for 4 h. The PDMS layer was then carefully peeled off and holes were punched in the 2 mm layer using a 0.5 mm biopsy punch to obtain the holes for the inlet and outlet. Both layers (2 mm and 1 mm thick) were irreversibly bonded by plasma activation, baked at 85 °C for 4 h, and punched using a 4 mm biopsy punch to create cell chamber holes, and the resulting 3 mm thick PDMS layer was irreversibly bonded to the thin PDMS layer on a glass slide and heated for 4 h at 85 °C. Finally, a second glass slide was used to cover the holes during the experiment.

[0169] RTA using microfluidics (present invention)

[0170] Place the chip in an oven and bake at 85°C for 1 hour. Add 10 μL of sterile MiliQ water containing 15 μg / mL poly-L-lysine to each well and incubate at 37°C for 40 minutes to cover the well surface, then wash with MiliQ water. Resuspend 100,000 cells in 600 μL of complete medium (the medium used in cell culture) and divide into 3 different test tubes, where the treatment agent is added at appropriate concentrations. Add 35 μL of cell suspension to each well and incubate the chip at 37°C for 16 hours. At the end of the incubation, refill the well with complete medium and seal it with a glass slide, which is fixed with a plastic crocodile clip. Then, use a P2SC pump to perfuse complete medium containing 100 nM TMRE and 2 μM calcein AM through two inlets at a pressure of 300 mbar for 15 minutes, and incubate the chip at 37°C for 40 minutes. Next, complete medium was perfused at 200 mbar pressure through one inlet for 20 minutes, while the same buffer containing 1 μM of the apoptosis catalyst combination of the present invention was perfused in the other inlet. The chip was incubated at room temperature for 2 hours, and then images were acquired using a Nikon fluorescence microscope.

[0171] Characterization of gradients

[0172] A MiliQ water solution containing 10 mg / mL bovine serum albumin (BSA) (A3059, Sigma-Aldrich) and 428 μL / mL blue food dye (Vahine, Catalonia, Spain) was perfused through the first inlet of the chip at 200 mbar using a precision pressure control system P2CS pump (Biopyhysical tools, Leipzig, Germany), while only MiliQ water was perfused through the second inlet. The liquid flowing out of the outlet was collected in an Eppendorf. The Pierce TM BSA concentration was measured with a BCA protein quantification kit (23225, Thermo Fisher Scientific) and absorbance at a blue wavelength of 640 nm was measured using a Benchmark Plus microplate reader (4100172C, Bio-Rad, CA, USA). Fluorescence gradients were characterized by injecting 10 mM NaOH containing 25 μg / mL fluorescein (F2456, Sigma-Aldrich) and capturing images using a 436 Zeiss Axio Observer Z1 / 7 microscope.

[0173] Statistical analysis

[0174] All results are presented as mean ± SEM of at least three biologically independent replicates. Each condition was compared with the control condition using an unpaired t-test, and p values ​​below 0.05 were marked as statistically significant (*). Statistical analysis was performed and the results are presented using GraphPad Prism 9.

[0175] result

[0176] Real-time tracking of apoptosis identifies effective treatments for liquid tumors

[0177] The inventors compared the efficiency of initiating cell apoptosis using the combination of the present invention with the method reported in the prior art WO2014047342 (hereinafter also referred to as "standard DBP").

[0178] In the NALM-6 cell line, RTA showed a dose-response curve similar to that of standard DBP, indicating that titration of the apoptosis catalyst combination of the present invention had a similar effect as the BIM peptide. Importantly, in cells treated with trametinib, lower concentrations of apoptosis catalysts and BIM peptides were required to initiate the apoptosis process, whereas in the case of imatinib, the same curve as the control condition was obtained ( Figure 1 A and Figure 1 C).

[0179] Similar results were obtained in SEM cells, where SEM cells treated with sunitinib were more sensitive to the apoptosis catalyst and BIM peptide of the present invention, showing increased apoptosis initiation associated with future cytotoxicity ( Figure 1 B and Figure 1 D).

[0180] Results from this type of assay can also be expressed as Δ% initiation, which represents the difference in apoptosis initiation between treatment and control conditions at a given concentration in the titration. Comparing the results from DBP and RTA, Δ% initiation increased after treatment with trametinib in NALM-6 and after treatment with sunitinib in SEM, whereas for both cell lines, apoptosis initiation did not increase after imatinib treatment ( Figure 1 E and 1F). These results suggest that the new RTA protocol can identify effective therapeutics in BCP-ALL cell lines with efficacy equal to or greater than that of standard DBP and has the significant advantage that cells do not require a permeabilization step with digitonin.

[0181] For solid tumors, RTA can be performed using flow cytometry and fluorescence microscopy.

[0182] To demonstrate that the new assay is also applicable to adherent cells, two gastrointestinal stromal tumor (GIST) cell lines were used, GIST-T1 and GIST-T1 / 670. As previously published, the GIST-T1 cell line is sensitive to imatinib treatment, whereas GIST-T1 / 670 is resistant to this inhibitor.

[0183] Likewise, standard DBP was performed on GIST-T1 after treatment with imatinib and gefitinib as a negative control. Pretreatment with imatinib resulted in a decrease in the concentration of BIM peptide required to initiate the apoptotic process compared to untreated conditions, whereas treatment with gefitinib showed a curve similar to that of the control ( Figure 2 A). For the GIST-T1 / 670 cell line, the three experimental conditions had the same dose-response curves, as both imatinib and gefitinib were ineffective against these cells ( Figure 2 B).

[0184] When RTA was performed using the apoptosis catalyst of the present invention and read by flow cytometry, a dose response curve was obtained. Similar to the results in BCP-ALL, treatment of GIST-T1 with imatinib resulted in a decrease in the concentration of apoptosis inducers that initiate the apoptotic process, showing an increase in apoptosis initiation after treatment with an effective drug ( Figure 2 C). Similarly, treatment with imatinib in the resistant cell line GIST-T1 / 670 or with gefitinib in both cell lines produced curves identical to those of the control condition ( Figure 2 D).

[0185] The new protocol provided by the present invention avoids the use of digitonin to permeabilize cells, but fixes the cells at the end of the incubation to allow immunostaining of cytochrome C. In order to improve it, a new method based on TMRE has been developed, which is a fluorescent dye that accumulates in healthy mitochondria (non-apoptotic cells) but loses fluorescence when mitochondria are permeabilized (apoptotic cells). In order to prove that this method using a microscope achieves similar results, RTA was adjusted to add this dye. The results showed that GIST-T1 cells provided a dose-response curve based on TMRE intensity that could distinguish apoptotic cells from non-apoptotic cells. Importantly, pretreatment with imatinib caused the curve to shift, thereby identifying an increase in the initiation of apoptosis, whereas this was not observed in cells treated with gefitinib (negative control) ( Figure 2 E). As expected, for GIST-T1 / 670 cells, both treatments produced unchanged curves, similar to the control condition ( Figure 2 F).

[0186] When Δ% initiation was quantified for all three methods, similar results were obtained in all cases, with a clear increase in apoptosis initiation after imatinib treatment for GIST-T1 cells, whereas it did not change significantly in the other conditions ( Figure 2 G and 2H). This shows that RTA can be performed on adherent cells using both flow cytometry and fluorescence microscopy, and in the latter case, the use of TMRE as a fluorescent marker for the induction of apoptosis obviates the need for an immunostaining step.

[0187] RTA works when using a combination of other apoptosis catalysts of the present invention

[0188] The first experiment focused on a cell apoptosis catalyst consisting of ABT-199, A1331852 and S63845, which was named Combination A.

[0189] To demonstrate that any combination of agents that simultaneously affect BCL-2, BCL-xL, and MCL-1 could be used to perform RTA, two new apoptosis catalysts were prepared.

[0190] Combination B was composed of three different direct inhibitors of the aforementioned anti-apoptotic proteins (S55746, WEHI-539, and AZD5991).

[0191] Combination C combines other direct inhibitor combinations (ABT-199 and AZD5991) and adds DT2216, a PROTAC drug that acts as an indirect inhibitor affecting BCL-xL by degrading BCL-xL.

[0192] The same experimental setup was first performed using the BCP-ALL cell line NALM-6 and SEM.

[0193] Flow cytometric RTA was performed in the NALM-6 cell line using combination B and combination C, and dose-response curves were obtained under untreated and imatinib-treated conditions, however, under trametinib-treated conditions, both combination B and combination C required less apoptotic catalysts to induce apoptosis ( Figure 3 A and Figure 3 C).

[0194] In SEM cells, the results were similar, showing that treatment with sunitinib induced the initiation of apoptosis compared to untreated cells, both with combination B and combination C ( Figure 3 B and Figure 3 D).

[0195] Finally, similar results were obtained when the Δ% initiation was compared between standard DBP and RTA using the three apoptosis catalyst combinations.

[0196] In all cases, targeting NALM-6 with trametinib and SEM with sunitinib induced and increased apoptosis initiation ( Figure 3 E and 3F).

[0197] Using adherent cell models (GIST-T1 and GIST-T1 / 670), RTA was performed using a novel combination of apoptosis promoters following anticancer treatment.

[0198] Combination B produced a dose-response curve for both cell lines, and the only treatment that altered the curve was imatinib in GIST-T1 cells, as expected ( Figure 4 A and 4B).

[0199] In the case of combination C, untreated cells did not respond to increasing concentrations of apoptotic activators, which may be due to the mechanism of action of DT2216 and the incubation time. However, in GIST-T1 cells treated with imatinib, combination C caused a shift in the dose-response curve, showing an induction of apoptosis initiation and identifying an effective drug ( Figure 4 C and 4D). Likewise, when comparing the Δ% initiation caused by the three combinations of the present invention and the standard DBP, the use of imatinib in GIST-T1 cells was the only case that showed an increase in apoptosis initiation ( Figure 4 E and 4F).

[0200] These results demonstrate that any combination of actions on the three major anti-apoptotic proteins (BCL-2, BCL-xL, and MCL-1), either through direct inhibition or by indirectly targeting the proteins, can be used to identify apoptosis induction for anticancer therapeutics.

[0201] RTA can be performed in microfluidic chips

[0202] One of the main limitations of the classic DBP and initial RTA experiments is that both require large numbers of living cells to perform the measurements. The inventors have integrated the combination of the present invention into a microfluidic chip, greatly reducing the number of cells required and allowing for automated processes, thus facilitating their implementation in the clinic.

[0203] GIST-T1 cells were seeded and treated within the microfluidic device, exposing the cells to gradient concentrations of BIM peptides generated directly by the device. As shown in the dose-response curve, increasing concentrations of BIM peptides induced the initiation of apoptosis in control cells. Lower concentrations of peptide were required for the same cells treated with imatinib, indicating an increase in the initiation of apoptosis ( Figure 5 A).

[0204] As described above, by using fluorescence microscopy, RTA using combination A was able to identify the induction of apoptosis in GIST-T1 cells after treatment with imatinib ( Figure 5 B). Using the same protocol within the microfluidic device, GIST-T1 cells were seeded and treated with imatinib and gefitinib (negative control). The device itself was used to titrate apoptotic catalysts, and dose response curves were generated using TMRE to identify apoptotic and non-apoptotic cells. Untreated GIST-T1 cells responded to Combination A in the same way as gefitinib-treated cells, but for imatinib-treated cells, fewer apoptotic catalysts were required to initiate the process ( Figure 5 C). When quantifying Δ% initiation, the microfluidic version of RTA was able to identify increased initiation of apoptosis caused by imatinib in GIST-T1 cells, similar to the microfluidics-based DBP and microscopy-based RTA ( Figure 5 D).

[0205] The microfluidic device enables the identification of effective therapeutics using a combination of BH3 mimetics as apoptosis catalyst combinations of the present invention, but using fewer cells. The combination of RTA and the microfluidic device can be further developed to generate a kit for identifying anticancer drugs in patient samples at the hospital site.

[0206] Terms

[0207] For reasons of completeness, various aspects of the invention are listed in the following numbered clauses:

[0208] Item 1. A combination comprising a BCL-2 inhibitor, a BCL-xL inhibitor and a MCL-1 inhibitor.

[0209] Clause 2. The combination according to clause 1, which consists of a BCL-2 inhibitor, a BCL-xL inhibitor and a MCL-1 inhibitor.

[0210] Clause 3. A combination according to any of the preceding clauses, comprising or consisting of ABT-199, A-1331852 and S63845.

[0211] Item 4. A kit comprising the inhibitor combination as defined in any one of items 1 to 3.

[0212] Item 5. The kit according to Item 4, which is a microfluidic device.

[0213] Item 6. Kit according to Item 5, comprising means for generating a gradient, in particular a logarithmic gradient, within the device.

[0214] Item 7. A kit according to any one of the preceding items 6 to 7, comprising a T-junction gradient generator.

[0215] Item 8. A combination according to any one of items 1 to 3 or a kit according to any one of items 4 to 7, comprising one or more additional anti-apoptotic BCL-2 family inhibitors having a molecular weight below 2 KDa, in particular below 1.5 KDa, in particular below 1 KDa.

[0216] Clause 9. A method for predicting the cytotoxicity of a therapeutic agent to a cell, the method comprising:

[0217] a) contacting the isolated test cell population with a test therapeutic agent;

[0218] b) contacting the cell population obtained in step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein the one or more anti-apoptotic BCL-2 family inhibitors have a molecular weight of less than 2 KDa, in particular less than 1.5 KDa, in particular less than 1 KDa;

[0219] c) determining the initiation value of cell apoptosis;

[0220] d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and

[0221] e) comparing the two start values;

[0222] in:

[0223] - step (d) is performed in the absence of a therapeutic agent;

[0224] - the process is performed in the absence of a cell permeabilizing agent; and

[0225] Therein, a decrease in the trigger value of the test cell fraction compared to the trigger value of the reference cell fraction indicates that the therapeutic agent is cytotoxic to the cells.

[0226] Clause 10. A method for predicting a cytoprotective effect of a therapeutic agent, the method comprising:

[0227] a) contacting the isolated test cell population with a test therapeutic agent;

[0228] b) contacting the cell population obtained in step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein the one or more anti-apoptotic BCL-2 family inhibitors have a molecular weight of less than 2 KDa, in particular less than 1.5 KDa, in particular less than 1 KDa;

[0229] c) determining the initiation value of cell apoptosis;

[0230] d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and

[0231] e) comparing the two start values;

[0232] in:

[0233] - step (d) is performed in the absence of a therapeutic agent;

[0234] - the process is performed in the absence of a cell permeabilizing agent; and

[0235] Therein, an increase in the trigger value of the test cell fraction compared to the trigger value of the reference cell fraction indicates that the therapeutic agent has a protective effect on the cells.

[0236] Clause 11. The method according to any one of clauses 9 to 10, wherein the triggering value is determined using different concentrations of the one or more anti-apoptotic BCL-2 family inhibitors, thereby obtaining respective triggering curves for the test sample and the reference sample.

[0237] Clause 12. A method according to clause 11, wherein:

[0238] When the launch curve of the test sample is shifted to the left relative to the launch curve of the reference sample, it indicates that the tested therapeutic agent is cytotoxic to the cells; and

[0239] When the activation curve of the test sample is shifted to the right relative to the activation curve of the reference sample, it indicates that the tested therapeutic agent has a protective effect on the cells.

[0240] Clause 13. The method according to any one of clauses 9 to 12, wherein steps (c) and (d) are performed by measuring the mitochondrial outer membrane permeability of the test cell population and the reference cell population.

[0241] Item 14. A method according to any one of items 9 to 13, wherein measuring the mitochondrial outer membrane permeability comprises measuring the release of molecules from the mitochondrial membrane space; in particular comprising the release of one or more of the following molecules from the mitochondrial membrane space: cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2 or apoptosis inducing factor (AIF); in particular the release of cytochrome c.

[0242] Clause 15. The method according to any one of clauses 9 to 14, wherein the cell population is a cancer cell population and the therapeutic agent is a chemotherapeutic agent; especially a targeted chemotherapeutic agent; especially a kinase inhibitor.

Claims

1. A non-therapeutic in vitro use of a combination of a BCL-2 inhibitor, a BCL-xL inhibitor and a MCL-1 inhibitor for initiating apoptotic state in an isolated test cell sample.

2. The in vitro use according to claim 1, wherein the combination consists of a BCL-2 inhibitor, a BCL-xL inhibitor and a MCL-1 inhibitor.

3. The in vitro use according to any one of the preceding claims, wherein the combination comprises direct inhibitors of BCL-2, MCL-1 and BCL-xL.

4. The in vitro use according to any one of the preceding claims, wherein the combination comprises a direct inhibitor of BCL-2 and MCL-1 and an indirect inhibitor of BCL-xL.

5. The in vitro use according to any one of the preceding claims, wherein the combination comprises or consists of one of the following: ABT-199, A-1331852, and S63845; ABT-199, DT2216, and AZD5991; and S55746, WEHI-539, and AZD5991.

6. A kit comprising the inhibitor combination as defined in any one of claims 1 to 5. The kit according to claim 6 , which is a microfluidic device.

8. Kit according to claim 7, comprising means for generating a gradient, in particular a logarithmic gradient, within the device.

9. A kit according to any one of the preceding claims 7 to 8, comprising a T-junction gradient generator.

10. The in vitro use according to any one of claims 1 to 5 or the kit according to any one of claims 6 to 9, wherein the combination further comprises one or more other anti-apoptotic BCL-2 family inhibitors with a molecular weight below 2 KDa, in particular below 1.5 KDa, in particular below 1 KDa.

11. A method for predicting a subject's response to treatment, the method comprising the steps of: (a) contacting a test cell sample isolated from the subject with a candidate drug; (b) initiating an apoptotic state in the cells by contacting the cell sample with a combination as defined in any one of claims 1 to 5; (c) Determine the trigger value of the test sample; (d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and (e) Compare the two starting values.

12. A method for predicting the cytotoxicity of a therapeutic agent to a cell, the method comprising: a) contacting the isolated test cell population with a test therapeutic agent; b) initiating the apoptotic state of the cells by contacting the cell population obtained in step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein the molecular weight of the one or more anti-apoptotic BCL-2 family inhibitors is lower than 2 KDa, especially lower than 1.5 KDa, especially lower than 1 KDa; c) determining the initiation value of cell apoptosis; d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with the one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and e) comparing the two start values; in: - step (d) is performed in the absence of a therapeutic agent; - the process is performed in the absence of a cell permeabilizing agent; and Wherein, a decrease in the trigger value of the test cell fraction compared to the trigger value of the reference cell fraction indicates that the therapeutic agent is cytotoxic to the cells.

13. A method for predicting the cytoprotective effect of a therapeutic agent, the method comprising: a) contacting the isolated test cell population with a test therapeutic agent; b) initiating the apoptotic state of the cells by contacting the cell population obtained in step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein the molecular weight of the one or more anti-apoptotic BCL-2 family inhibitors is lower than 2 KDa, especially lower than 1.5 KDa, especially lower than 1 KDa; c) determining the initiation value of cell apoptosis; d) obtaining a reference apoptosis initiation value by contacting another isolated cell population sample from the subject with the one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); and e) comparing the two start values; in: - step (d) is performed in the absence of a therapeutic agent; - the process is performed in the absence of a cell permeabilizing agent; and Wherein, an increase in the trigger value of the test cell fraction compared to the trigger value of the reference cell fraction indicates that the therapeutic agent has a protective effect on the cells.

14. The method according to any one of claims 11 to 13, wherein the triggering value is determined using different increasing concentrations of the one or more anti-apoptotic BCL-2 family inhibitors, thereby obtaining respective triggering curves for the test sample and the reference sample.

15. The method according to claim 14, wherein: When the activation curve of the test sample is shifted to the left relative to the activation curve of the reference sample, it indicates that the tested therapeutic agent is cytotoxic to cells; as well as When the activation curve of the test sample shifts to the right relative to the activation curve of the reference sample, it indicates that the tested therapeutic agent has a protective effect on cells.

16. The method according to any one of claims 11 to 15, wherein steps (c) and (d) are performed by measuring the mitochondrial outer membrane permeability of the test cell population and the reference cell population.

17. The method according to any one of claims 11 to 16, wherein measuring the mitochondrial outer membrane permeability comprises measuring the release of molecules from the mitochondrial intermembrane space; in particular comprising the release of one or more of the following molecules from the mitochondrial intermembrane space: cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2 or apoptosis inducing factor (AIF); in particular the release of cytochrome c.

18. The method according to any one of claims 11 to 17, wherein the cell population is a cancer cell population and the therapeutic agent is a chemotherapeutic agent; in particular a targeted chemotherapeutic agent; in particular a kinase inhibitor.

19. The method according to any one of claims 11 to 18, which is performed using a combination or a kit as defined in any one of claims 1 to 10.

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