Real-time tracking of apoptosis

Abstract

The present invention provides compositions, kits and methods that allow prediction of the cytotoxicity or cytoprotection of therapeutic agents without altering the permeability of the cells tested.

Description

[Technical Field]

[0001] The present invention relates to predicting the efficacy of anti-cancer treatment. More specifically, the present invention provides a combination of anti-apoptotic inhibitors useful for predicting the sensitivity of a subject to anti-cancer therapy, as well as a kit and device comprising the same. [Background technology]

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

[0003] In recent years, the development of tyrosine kinase inhibitors (TKIs) has improved the treatment of patients with advanced disease. For example, detection of mutations in EGFR has been successfully used as a biomarker for initial treatment with EGFR inhibitors. However, many targeted agents lack genetic predictive markers. Furthermore, resistance to these drugs often emerges, and given the various mechanisms for resistance, it is often unclear what treatment is best given after this resistance emerges.

[0004] With the emergence of precision medicine in cancer, there is a dire need for new predictive biomarkers to assess the efficacy of treatment in patients. Beyond standard molecular analyses, functional assays are now being developed to guide clinical decisions.

[0005] In this regard, the use of so-called dynamic BH3 profiling or DBP to directly evaluate treatments has been reported.

[0006] This method is based on catalyzing the process of apoptotic cell death by using synthetic BH3 peptides. However, the actual method reported in this paper requires permeabilization of cells with digitonin to allow internalization of the BH3 peptides and implement their pro-apoptotic activity. This represents a clear limitation of the assay, as it forces cells to be fixed (and therefore killed) after incubation with the peptide, which allows visualization of only one time point. See, for example, US Patent No. 20220163510 or US Patent No. 10393733.

[0007] Therefore, despite efforts, there remains a need for additional tools to adequately predict response to anticancer drugs. Summary of the Invention

[0008] The present inventors have identified combinations of anti-apoptotic inhibitors that can provide efficient and sensitive predictive information regarding a subject's response to a particular treatment or treatments.

[0009] In particular, the present inventors have designed a molecular combination that provides inhibitory effects against BCL-2, BCL-xL, and MCL-1. As shown below, the inhibitors forming the combination of the present invention can efficiently penetrate cancer cells without damaging them. Therefore, no permeabilizing agent is required.

[0010] Furthermore, the inventors have found that the apoptotic state induced by the combination of inhibitors of the present invention is at least equal to or even greater than the apoptotic effect provided by the DBP protocol provided in the prior art.

[0011] Thus, the present invention provides a combination that is at least as efficient as the BH3 peptide / digitonin combination already reported in prior art DBP protocols in determining apoptotic priming, with the notable advantage that cell permeabilization is not required in the present case, and therefore cell viability of the test cell population sample is not adversely affected.

[0012] The above represents a significant advance over DBP methodologies disclosed in the prior art to date.

[0013] In contrast, the prior art required two steps to achieve the apoptosis priming effect: (1) increasing the permeability of the cell membrane by administering digitonin, and (2) contacting the cells with a synthetic BH3 peptide to catalyze apoptosis.

[0014] Advantageously, the combination of the present invention avoids the permeabilization step and allows for continuous monitoring of live cells.

[0015] On the other hand, one of the major concerns in cancer management is not only predicting the response when a subject is intended to start anticancer therapy, but also how the subject may respond to long-term anticancer therapy (e.g., the development of resistance to treatment).

[0016] A key advantage of using the inhibitor combinations of the present invention (also referred to as "apoptosis catalyst combinations") is that when administered, the combinations can prime a highly apoptotic state without adversely affecting the integrity of cancer cells. This allows for real-time monitoring of the evolution of cancer cells over long periods of treatment with a single test sample. This information is extremely useful in the appropriate management of different stages of cancer disease, predicting the likelihood of developing resistance to treatment, and testing several anticancer therapies over time.

[0017] Overall, the combinations provided by the present invention represent a major advance in the efficient 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 an MCL-1 inhibitor.

[0019] In a second aspect, the present invention provides a kit comprising a combination of inhibitors as defined in the first aspect of the invention.

[0020] The present inventors have developed a method capable of predicting a subject's sensitivity to a particular candidate drug(s) based on one or more anti-apoptotic BCL-2 family inhibitors having a molecular weight of less than 2.0 KDa.

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

[0022] The methods of the present invention are based on measuring how close cells are to the threshold for programmed cell death (i.e., apoptosis), also known as measuring how "primed" cancer cells are to death. The methods of the present invention allow for the identification of drugs that bring cells closer to the threshold for programmed cell death (the drugs that most increase apoptotic priming). These methods of the present invention can be applied to individual clinical cancer samples, thereby readily identifying drugs that move cells in that sample closest to the threshold for programmed cell death for that individual sample. Drugs so identified are those most likely to provide clinical benefit to the subject from whom the sample was derived. Thus, the present invention provides a method for personalizing treatment for individual cancer patients based on the use of one or more small molecule inhibitors of the BCL-2 anti-apoptotic family.

[0023] Thus, 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; and (b) contacting the cell population resulting from step (a) with a particular concentration of 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, particularly less than 1.5 KDa, and particularly less than 1 KDa; (c) determining an apoptotic priming value; (d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from the subject with the one or more anti-apoptotic BCL-2 family inhibitors used in step (b); and (e) comparing both priming values, wherein step (d) is performed in the absence of the therapeutic agent and the process is performed in the absence of a cell permeabilizing agent; a decrease in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent is cytotoxic to the cells. The resulting change in apoptotic priming between treated and untreated cells after exposure to BCL-2 family inhibitor(s) results in a change in apoptotic priming upon treatment (usually defined as Δ% priming), which indicates the ability of the treatment to engage in apoptosis and eliminate target cells. Because the present invention allows this determination to be performed on live cancer cells, several treatments can be administered and monitored over time as a real-time tracker of apoptotic engagement.

[0024] In a fourth aspect, the present invention provides a method for predicting the cytoprotection of a therapeutic agent, the method comprising: (a) contacting an isolated test cell population with a test therapeutic agent; (b) contacting the cell population resulting from step (a) with a particular concentration of 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, particularly less than 1.5 KDa, and particularly less than 1 KDa; and (c) determining whether the cell population resulting from step (a) is cytoprotective. (d) determining a reference apoptosis priming 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 both priming values, wherein step (d) is performed in the absence of the therapeutic agent and the process is performed in the absence of a cell permeabilizing agent; and an increase in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent protects the cells. [Brief explanation of the drawings]

[0025] [Figure 1]Percentage of cytochrome c retained (Y-axis) in A) NALM-6 and B) SEM cell lines after 16 hours of treatment with anti-cancer drugs and exposure to different concentrations of BIM peptide (X-axis; for comparison, standard DBP). Percentage of cytochrome c retained (Y-axis) in C) NALM-6 and D) SEM cell lines after 16 hours of treatment with anti-cancer drugs and exposure to different concentrations of Combination A of the present invention (X-axis) as an apoptosis catalytic agent, using flow cytometry. Comparison of Δ% priming between normal DBP, cytometric RTA, and microscopic RTA using an apoptosis catalytic agent in E) NALM-6 and F) SEM cell lines. Squares = reference; circles = trametinib 100 nM in (A) and (C); or sunitinib 1000 nM in (B) and (D); triangles = imatinib 1000 nM. In (E) and (F), black bars represent Δ% priming by DBP, and white bars represent Δ% priming by cytometric RTA. [Figure 2]Percentage of cytochrome c retained (Y-axis) in A) GIST-T1 and B) GIST-T1 / 670 cell lines after 16 hours of treatment with anticancer drugs and exposure to different concentrations of BIM peptide (X-axis; for comparison, standard DBP). Percentage of cytochrome c retained (Y-axis) in C) GIST-T1 and D) GIST-T1 / 670 cell lines after 16 hours of treatment with anticancer drugs and exposure to different concentrations of Combination A of the present invention as an apoptosis catalyst (X-axis) using flow cytometry. Percentage of TMRE-positive cells (Y-axis) in E) GIST-T1 and F) GIST-T1 / 670 cell lines after 16 hours of treatment with anticancer drugs and exposure to different concentrations of Combination A of the present invention as an apoptosis catalyst (X-axis) using fluorescence microscopy. Comparison of Δ% priming between normal DBP, cytometric RTA, and microscopic RTA using apoptosis-catalyzing agents in GIST-T1 and H) GIST-T1 / 670 cell lines. Squares = reference; Circles = imatinib 1000 nM; Triangles = gefitinib 1000 nM. In (E) and (F), black bars represent Δ% priming with DBP, gray bars represent Δ% priming with cytometric RTA, and white bars represent Δ% priming with microscopic RTA. [Figure 3]Percentage of cytochrome c retained (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of inventive combination B (X-axis) as apoptosis catalytic agents in A) NALM-6 cell line and B) SEM cell line using flow cytometry. Percentage of cytochrome c retained (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of inventive combination C (X-axis) as apoptosis catalytic agents in C) NALM-6 cell line and D) SEM cell line using flow cytometry. Comparison of Δ% priming between cytometric RTA using normal DBP, combination A (from Figure 1), combinations B and C in E) NALM-6 cell line and F) SEM cell line. Squares = reference; circles = trametinib 100 nM in (A) and (C); or sunitinib 1000 nM in (B) and (D); triangles = imatinib 1000 nM. Black bars represent Δ% priming by DBP in (E) and (F), dark gray bars represent RTA using combination A, light gray bars represent RTA using combination B, and white bars represent RTA using combination C. [Figure 4]Percentage of cytochrome c retained (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of inventive combination B (X-axis) as apoptosis catalyst in A) GIST-T1 and B) GIST-T1 / 670 cell lines using flow cytometry. Percentage of cytochrome c retained (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of inventive combination C (X-axis) as apoptosis catalyst in C) GIST-T1 and D) GIST-T1 / 670 cell lines using flow cytometry. Comparison of Δ% priming between cytometric RTA using normal DBP, combination A (from Figure 1), combinations B and C in E) GIST-T1 and F) GIST-T1 / 670 cell lines. Squares = reference; circles = imatinib 1000 nM; triangles = gefitinib 1000 nM. Black bars represent Δ% priming by DBP in (E) and (F), dark gray bars represent RTA using combination A, light gray bars represent RTA using combination B, and white bars represent RTA using combination C. [Figure 5]A) The percentage of TMRE-positive cells (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of BIM peptide (X-axis) using a microfluidic platform and fluorescence microscopy in the GIST-T1 cell line. B) The percentage of cytochrome c retained after 16 hours of treatment with anticancer drugs and exposure to different concentrations of Combination A of the present invention (X-axis) as an apoptosis catalyst in the GIST-T1 cell line using flow cytometry (Y-axis). C) The percentage of TMRE-positive cells (Y-axis) after 16 hours of treatment with anticancer drugs and exposure to different concentrations of Combination A of the present invention (X-axis) as an apoptosis catalyst in the GIST-T1 cell line using a microfluidic platform and fluorescence microscopy. D) Comparison of Δ% priming between normal DBP in a microfluidic chip, microscopic RTA with Combination A in a microfluidic chip, and RTA with Combination A. Squares = reference; circles = imatinib 1000 nM; triangles = gefitinib 1000 nM. Black bars represent Δ% priming with DBP inside the microfluidic chip, grey bars represent microscopic RTA using combination A, and white bars represent RTA using combination A inside the microfluidic chip. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description All terms used herein in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are as follows, and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0027] For purposes of the present invention, any range given includes both the lower and upper endpoints of the range.

[0028] As used herein, the meaning of the term "comprising" encompasses three options: "comprising," "consisting of," and "consisting essentially of."

[0029] In a first aspect, the present invention provides a combination comprising molecules that inhibit BCL-2, BCL-xL and MCL-1.

[0030] In the context of the present invention, the term "inhibitor" encompasses both molecules that have a direct effect on a target (e.g., by binding; direct inhibitors) and molecules that trigger mechanisms that provide an inhibitory or downregulatory effect on BCL-2, BCL-xL, and MCL-1 (indirect inhibitors). As demonstrated below, whether a direct or indirect inhibitor is involved, the relevant technical feature is that the combination must inhibit BCL-2, BCL-xL, and MCL-1; this simultaneous inhibition is an essential feature for inducing an apoptotic state and determining apoptotic priming, as explained above.

[0031] Those skilled in the art know how to determine whether a molecule is an inhibitor of any of these targets, and many publications have already reported the identification and use of molecules that inhibit BCL-2, BCL-xL, and MCL-1.

[0032] In the context of the present invention, a combination can include one molecule specific for each target, i.e., a combination includes three molecules. In other cases, a combination can include a molecule that exerts a dual effect on two targets (e.g., a BCL-2 / BCL-xL inhibitor). In this alternative case, the combination includes two molecules for inhibiting BCL-2, BCL-xL, and MCL-1, one with a dual effect and a second molecule specific for the other target.

[0033] The intrinsic pathway, also known as the mitochondrial pathway, is regulated by the BCL-2 family of proteins, which has distinct members that can be classified based on structure, function, and BCL-2 homology (BH) domains: activating members, antiapoptotic members, sensitizers, and effectors.

[0034] In the context of the present invention, the molecules incorporated into the combination are those that bind with high affinity and specificity to the hydrophobic groove of anti-apoptotic protein targets and inhibit them.

[0035] There are known inhibitors of each of the three families (BCL-2, BCL-xL and MCL-1), some of which are detailed below by way of example (see also Table 1).

[0036] Molecules that directly inhibit BCL-2 The first molecule described to target BCL-2 was HA14-1, which showed in vitro and in vivo activity alone or in combination with cytotoxic therapy.

[0037] Abbott Laboratories (now AbbVie) developed ABT-737, the first on-target specific BH3 mimetic that blocks the BH3 binding domain and inhibits BCL-2, BCL-xL, and BCL-W. This compound was further purified to improve its oral bioavailability, resulting in the analog ABT-263 (navitoclax).

[0038] Navitoclax has shown promising results in several blood cancers, particularly chronic lymphocytic leukemia (CLL), which expresses high levels of BCL-2.

[0039] To reduce the risk of thrombocytopenia, AbbVie developed a selective BCL-2 inhibitor called ABT-199 (venetoclax). Clinical responses of CLL patients to venetoclax have been evidence of impressive anticancer activity, resulting in rapid weight loss and even cases of tumor lysis syndrome.

[0040] Other illustrative non-limiting examples of BCL-2 inhibitors are APG-1252, BGB-11417, S55746 or SPC2996, among others.

[0041] The examples provided below are based on combinations that include either ABT-199 or S55746 as the BCL-2 inhibitor. The same results as those provided by the chemically unrelated ABT-199 or S55746 can also be obtained with any of the above-cited inhibitors from the same family, since they act on the same target and are small enough to diffuse through the cell membrane.

[0042] Molecules that directly inhibit BCL-xL Selective BCL-xL inhibitors are also being developed for their potential anticancer activity. One of the first compounds shown to be highly selective was WEHI-539, which was also effective against solid tumors. More recent derivatives of this first BCL-xL inhibitor, such as A-1155463 or A-1331852, have also been reported.

[0043] More recent derivatives of this first BCL-xL inhibitor, such as A-1155463 or A-1331852, are also being investigated with promising preclinical results.

[0044] The latter, in particular, is very promising due to its oral bioavailability. In contrast, a new strategy targeting BCL-xL degradation using a proteolysis-targeting chimera (PROTAC; DT2216), which has the inherent selectivity of targeting this anti-apoptotic protein in tumor cells rather than platelets, is currently under evaluation, has clinical potential, and will form part of the scope of this invention.

[0045] The examples provided below are based on combinations that include either A-1331852 or WEHI-539 as a BCL-xL inhibitor. Also, the same results as those provided by including A-1331852 or WEHI-539 could be obtained with any of the above-cited inhibitors from the same family, since they act on the same target and, due to their very small size, can diffuse effortlessly through cell membranes.

[0046] Molecules that directly inhibit MCL-1 The observation that the MCL-1 anti-apoptotic protein is commonly used by cancer cells to evade apoptosis has also stimulated the development of novel targeted therapies.

[0047] One of the first selective inhibitors, A-1210477, has shown excellent in vitro results in hematological malignancies and solid tumors, such as breast and lung cancer cell lines, particularly in combination with navitoclax. Encouraged by the anticancer activity of these molecules, a new generation of promising small molecule MCL-1 antagonists is currently in clinical development. These include, among others, S64315 / MIK665, AZD-5991, PRT1419, and AMG-176, which are currently being investigated in clinical trials (e.g., NCT02992483, NCT04629443, NCT03013998, NCT02675452, NCT04178902, NCT04543305), primarily in hematological malignancies. Other compounds that inhibit or degrade MCL-1 have also demonstrated activity in preclinical models. Given that MCL-1 is a short-lived protein, indirect targeting with CDK9 inhibitors such as alvocidib, AZD4573 (NCT03263637), or voruciclib (NCT03547115), or protein-selective degradation approaches have emerged as alternative strategies. BFL-1 / A1 can also be similarly targeted using CDK9 inhibitors or dual inhibitors of MCL-1 and BFL-1.

[0048] S63845 has also been reported as a highly selective MCL-1 inhibitor with no cross-reactivity to BCL-2 or BCL-xL.

[0049] The examples provided below are based on combinations including either S63845 or AZD5991 as the MCL-1 inhibitor. Similar results may be obtained with any of the above small molecule inhibitors, as they are also able to diffuse through the cell membrane.

[0050] Indirect targeting of anti-apoptotic BCL-2 proteins Anti-apoptotic proteins can also be disrupted using molecules that are not capable of directly inhibiting the BH3-binding domains of BCL-2, BCL-xL, and MCL-1. In this regard, various strategies have been developed to target these proteins.

[0051] For example, small molecules that directly activate the effector BAX (including BTSA1, BAM7, or BAI1) indirectly block all anti-apoptotic proteins.

[0052] The use of antisense oligonucleotides (eg, BP1002, G3139, or oblimersen sodium) blocks or alters mRNAs encoding anti-apoptotic proteins, impairing their synthesis.

[0053] PNT2258 is a 24-base DNA oligodeoxynucleotide specific for the BCL-2 promoter, which caused downregulation of the mRNA encoding BCL-2 and consequently reduced BCL-2 levels.

[0054] Drugs that inhibit other proteins can indirectly alter specific pathways, leading to changes in the expression of anti-apoptotic proteins, including, but not limited to, inhibition of CDK9 by dinaciclib, AZD4573, or alvocidib, which reduces MCL-1, or by mifepristone, a steroidal antiprogestogen that reduces BCL-2 expression.

[0055] The use of PROTAC constructs such as DT2216, which mark BCL-xL for degradation by the proteasome (thus reducing levels of the protein). [Table 1] TIFF2025526286000002.tif251170TIFF2025526286000003.tif49170

[0056] Those skilled in the art can easily use their general knowledge to select inhibitors from each of the three groups, or use promiscuous anti-apoptotic inhibitors, which are small molecules that inhibit several anti-apoptotic proteins at once (e.g., ABT-263, APG-1252, AZD0466, etc.), and combine them to obtain similar results.

[0057] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the BCL-2 inhibitor is ABT-199 or S55746.

[0058] 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.

[0059] 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.

[0060] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the inhibitors of BCL-2, BCL-xL and MCL-1 are direct inhibitors.

[0061] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the inhibitors of BCL-2 and MCL-1 are direct inhibitors and the inhibitor of BCL-xL is an indirect inhibitor.

[0062] In one embodiment of the first aspect of the invention, the combination comprises ABT-199, A-1331852 and S63845, or comprises S55746, WEHI-539 and AZD5991, or comprises ABT-199, DT2216 and AZD5991, optionally in combination with any of the embodiments provided above or below.

[0063] 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 only ABT-199, A-1331852 and S63845, or only S55746, WEHI-539 and AZD5991, or only ABT-199, DT2216 and AZD5991.

[0064] In another embodiment of the first 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 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, such as BCL-2, BCL-xL, BCL-W and MCL-1. These additional inhibitors have a molecular weight of less than 2 KDa, more particularly 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.

[0065] Activator proteins (BIM, BID, and PUMA) bind to and directly activate the central effectors of apoptosis, BAX and / or BAK1, which then undergo a conformational change to oligomerize and form pores in the mitochondrial membrane, triggering MOMP.

[0066] The function of sensitizer proteins (BAD, PMA-inducible protein 1 (commonly called 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 can 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 the activator or sensitizer as well as BAX and BAK1. The BH3-only proteins PUMA, NOXA, BID, and BIM have strong affinity for and inhibit MCL-1, whereas BAD has stronger affinity for BCL-2, BCL-W, and BCL-xL than for MCL-1.

[0067] In the context of the present invention, an "anti-apoptotic BCL-2 family inhibitor" is understood to mean any well-known small molecule that directly (by binding to BCL-2) or indirectly negatively affects BCL-2 cellular activity, either by eliminating / degrading BCL-2 or by binding and inhibiting its activity even if it is produced. In another embodiment of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the combination comprises one or more types of anti-apoptotic BCL-2 family inhibitors other than those belonging to the BCL-2 inhibitors, BCL-xL inhibitors, and / or MCL-1 inhibitors described in claim 1. The preparation of the combination is routinely carried out by mixing the small molecules under stirring. These molecules must be added at a concentration that provides an inhibitory effect on the target. This concentration usually corresponds to the concentration recommended by the manufacturer. However, those skilled in the art can easily adjust the concentration of a particular inhibitory molecule precisely, if necessary, by simply using routine testing, for example, based on the Ki value.

[0068] Examples of anti-apoptotic BCL-2 family inhibitors include HA14-2, obatoclax, 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-176, AMG-397, AZD-5991, PRT1419, S64315 / MIK665, S63845, MIM1, VU661013, GDC-0941, and the like.

[0069] The inhibitors forming part of the combination are included in amounts sufficient to provide an inhibitory effect. Those skilled in the art will know and can readily adjust the amount of each inhibitor required.

[0070] kit All embodiments provided above under the first aspect of the invention relating to combinations of inhibitors are also embodiments of the kit of the second aspect of the invention.

[0071] 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.

[0072] 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 as referred to above, e.g., activators of pro-apoptotic members of the BCL-2 family, e.g., 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, and other inhibitors and activators of the BCL-2 family. Illustrative examples are provided above.

[0073] 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 of the inhibitors in a separate compartment with 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 combination of inhibitors of the first aspect of the invention.

[0074] In another embodiment of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit is a microfluidic device.

[0075] Microfluidic devices are easy to fabricate, low-cost, and robust, enabling long-term use for routinely conducting predictive cancer treatment response assays in hospitals at minimal cost. Furthermore, because the cell requirements are lower than current technologies, these analyses can be performed on non-surgically obtained biopsies, such as fine-needle aspirates. From a clinical perspective, this approach could represent a breakthrough in how we treat cancer patients, significantly improving clinical outcomes even for the most complex cases (recurrent / refractory / metastatic tumors).

[0076] Any commercially available microfluidic device can be used in connection with the present invention simply by following the manufacturer's instructions.

[0077] Chemical gradients play an important role in many biological processes and regulate several cellular functions in vivo. Indeed, several examples of gradient-dependent phenomena exist in nature, such as chemotaxis, i.e., cell migration driven by the establishment of a gradient. Furthermore, chemical gradients have been shown to influence various cellular behaviors, such as tumorigenesis.

[0078] Therefore, studying and potentially reproducing these conditions in vitro is clearly important in life science related research.

[0079] In one embodiment of the second aspect of the invention, the kit is a microfluidic device comprising means for generating a logarithmic or linear gradient, in particular a logarithmic gradient, of the combination of inhibitors of the invention, thereby allowing replication of dose curves.

[0080] The two most common methods of creating chemical gradients in microfluidics are (i) the use of ad-hoc designed microchannels with at least two inlets where adjacent streams containing different concentrations of chemicals of interest (i.e., inhibitor combinations as defined in the first aspect of the present invention) are mixed to generate a gradient, and (ii) the use of the ability of cells to secrete and consume biochemicals in their vicinity in combination with slow perfusion to create a gradient in a direction parallel to the direction of fluid flow.

[0081] Those skilled in the art can already select the most appropriate one from among commercially available devices with gradient functions. If one wants to design their own device with a gradient, they can use general knowledge to select the more appropriate means for generating such a gradient from among different means. In one embodiment, optionally in combination with any of the embodiments provided above or below, the microfluidic device includes a gradient generator, in particular a T-junction gradient generator.

[0082] In this embodiment, the T-junction at the end of the microchannel is the convection unit. To separate convection through the side channel from diffusion across the main channel, the bulk flow rates through the inlet and outlet must be matched. Assuming one convection unit carries a constant solute concentration and the other unit carries a buffer solution, this allows for the creation of a gradient across the microchannel that remains constant as long as the solute flow and concentration in each convection unit remain the same.

[0083] We validated this device in cell lines and in patient tumor samples from human gastrointestinal stromal tumors (GISTs), a genomic-driven neoplasm currently treated with small-molecule inhibitors targeting the KIT and PDGFRA receptor tyrosine kinases. This novel technological approach enables noninvasive, rapid, and accessible evaluation of treatments directly on cells isolated from patients (even from nonsurgically obtained biopsies) in an accurate and reproducible manner. Furthermore, by fully integrating this assay within a microfluidic chip, these analyses can be standardized to require minimal handling, enabling their future use as routine assays in hospitals.

[0084] Furthermore, the versatility of this microchip will enable tumor monitoring in patients to adapt treatment strategies throughout disease progression.

[0085] This novel methodology facilitates precision medicine to overcome cancer resistance to therapy and avoid recurrence by enabling clinical implementation of functional assays for ongoing personalized cancer treatment.

[0086] Because the equipment required to perform this assay requires only a simple microfluidic pump and a fluorescent readout, this new technology can be easily incorporated into ready-to-use medical devices for in situ analysis in hospitals.

[0087] method In a third aspect, the present invention provides a method for predicting the cytotoxicity of a therapeutic agent to a cell.

[0088] In a fourth aspect, the present invention provides a method for predicting the cytoprotection of a therapeutic agent on a cell.

[0089] All embodiments provided above under the first or second aspect of the invention are also embodiments of the methods of the third and fourth aspects of the invention.

[0090] In a first step, the methods of the invention involve contacting an isolated test cell population with a test therapeutic agent.

[0091] A test cell population is isolated from a subject using any conventional technique and cultured using a well-recognized, standardized culture medium.

[0092] Because the cell requirements are lower than current techniques, the methods of the present invention can also be performed on non-surgically obtained biopsies such as fine needle aspirates. From a clinical perspective, this approach could represent a breakthrough in how we treat cancer patients, significantly improving clinical outcomes in even the most complex cases.

[0093] In one embodiment of the method of the third and fourth aspects of the present invention, optionally in combination with any of the embodiments provided above or below, the cells are derived from a subject who is known to have cancer or suspected to have cancer. The subject is preferably a mammal. The mammal may be, for example, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the subject has previously been diagnosed with cancer and may have already been treated for cancer. Alternatively, the subject has not previously been diagnosed with cancer.

[0094] The test cell population is then exposed to a therapeutic agent.

[0095] In one embodiment of the method of the third and fourth aspects of the invention, optionally in combination with any of the embodiments provided above or below, the therapeutic agent induces apoptosis. In one embodiment of the method of the third and fourth aspects of the invention, optionally in combination with any of the embodiments provided above or below, the therapeutic agent is a chemotherapeutic agent. In another embodiment of the method of the third and fourth aspects of the invention, optionally in combination with any of the embodiments provided above or below, the therapeutic agent is a targeted chemotherapeutic agent. In another embodiment of the method of the third and fourth aspects of the invention, optionally in combination with any of the embodiments 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 kinases such as BCR-ABL, ABL, and KIT. In another embodiment of this method, the other therapeutic agent is a chemotherapeutic agent such as doxorubicin, cisplatin, 5-FU, and other cytotoxic agents used as anticancer agents. In another embodiment of this method, the other therapeutic agent is an antibody, including immune checkpoint inhibitors such as pembrolizumab, rituximab, and trastuzumab. 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.

[0096] Next, the test cell population that has already been exposed to the test therapeutic agent is contacted with one or more of the BCL-2 family apoptosis inhibitors mentioned and specified in the previous embodiment.This step is carried out by using the conventional means in the state of the art, using the experimental conditions that are well established or can be easily optimized by those skilled in the art.An exemplary method is provided below in the example section, in which cell is contacted with the combination at 37 ℃ for an appropriate period of time to ensure the induction of apoptosis.

[0097] As explained above, in the context of the present invention, the term "anti-apoptotic BCL-2 family inhibitor" encompasses any small molecule (having a molecular weight of less than 1.9 KDa, 1.8 KDa, 1.7 KDa, 1.6 KDa, 1.5 KDa, 1.4 KDa, 1.3 KDa, 1.2 KDa, 1.1 KDa, or 1.0 KDa) that directly or indirectly inhibits the function of BCL-2 or induces its elimination. Potential targets include the pro-apoptotic members BAX, BAK1, BIM, BID, and BBC3 (commonly referred to as PUMA), 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 to and directly activate BAX and / or BAK1, the central effectors of apoptosis, which then undergo a conformational change to oligomerize and form pores in the mitochondrial membrane, triggering MOMP. Sensitizer proteins (BAD, PMA-inducible protein 1 (commonly called NOXA), BIK, BMF, and HRK) function to inhibit antiapoptotic proteins and / or displace activators (such as BIM or BAX or BAK1 monomers) that can ultimately lead to apoptosis. Antiapoptotic members of the BCL-2 family possess four BCL-2 homology domains that form a binding groove that sequesters the activator or sensitizer, as well as BAX and BAK1. The BH3-only proteins PUMA, NOXA, BID, and BIM have strong affinity for and inhibit MCL-1, whereas BAD has a stronger affinity for BCL-2, BCL-W, and BCL-xL than for MCL-1.

[0098] There are known molecules that modulate the above targets, including, in addition to those already mentioned above, HA14-2, obatoclax, 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-176, AMG-397, AZD-5991, PRT1419, S64315 / MIK665, S63845, MIM1, VU661013, GDC-0941, etc.

[0099] In one embodiment of the methods of the third and fourth aspects of the invention, optionally in combination with any of the embodiments provided above or below, priming values are determined at different increasing concentrations of one or more anti-apoptotic BCL-2 family inhibitors, thus obtaining priming curves (dose response) for the test sample and the reference sample. In this embodiment, - if the priming curve of the test sample is shifted to the left relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent is cytotoxic to the cells; If the priming curve of the test sample is shifted to the right relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent protects the cells.

[0100] In one embodiment of step (b), the cell population is contacted with the first aspect of the invention or a combination as defined in any of the embodiments provided above or below.

[0101] Step (c) of the method of the third or fourth aspect of the present invention determines apoptotic cell death in the sample. There are many well-established protocols known to those skilled in the art that allow determining the degree of apoptosis in cells (Banfalvi G. "Methods to detect apoptotic cell death", Apoptosis, 2017 February;22(2):306-323), most of which are based on detecting abnormalities in cell membranes (e.g., plasma membranes or mitochondrial membranes).

[0102] In one embodiment of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (c) comprises or consists of measuring mitochondrial outer membrane permeabilization in the test cell population.

[0103] Outer membrane permeabilization can be measured by many methods, for example, by the loss of mitochondrial membrane potential.The loss of mitochondrial membrane potential can be measured, for example, by measuring the release of molecules 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).

[0104] The release of molecules from mitochondrial intermembrane space can be measured by methods known in the art.For example, by using antibodies against the molecules to be measured, i.e., cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2 or apoptosis-inducing factor (AIF).Detection can be, for example, by ELISA, FACS, immunoblotting, immunofluorescence or immunohistochemistry.

[0105] In one embodiment of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (c) comprises or consists of determining the % of cytochrome C released from mitochondria.

[0106] In addition to measuring molecules released from the mitochondrial space, other intracellular and extracellular markers can be measured, allowing for the ability to distinguish subpopulations of cells.

[0107] Alternatively, outer membrane permeabilization can be determined by treating the cells with a potentiometric or radiometric dye prior to step (c).

[0108] Advantageously, titration curves can be generated comparing treated and reference cells to assess the apoptosis-inducing ability of a treatment.

[0109] In view of the above, in one embodiment of the third aspect of the invention, optionally in combination with any of the embodiments provided above or below, prior to step (c), the cell population is first contacted with a potentiometric or radiometric dye. Illustrative, non-limiting examples of potentiometric dyes are hydrorhodamine 123, or tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE), and in particular, tetramethylylrhodamine (TMRE).

[0110] JC-1 is a lipophilic cationic dye that enters mitochondria in proportion to the potential across the inner mitochondrial membrane. JC-1 exists as a monomer at low membrane concentrations. However, JC-1 accumulates in the mitochondrial matrix under conditions of higher mitochondrial potential. At these higher concentrations, JC-1 forms red fluorescent "J-aggregates." As a monomer, the dye has an absorption / emission maximum at 527 nm, but at high membrane potentials, the emission maximum is 590 nm. Therefore, ratiometric measurements of this cyanine dye's emission can be used as a sensitive measure of mitochondrial membrane potential. The dye allows for dual measurements of dye concentration without the need for nuclear or cytoplasmic reference measurements. Studies using isolated mitochondria have shown that 527 nm emission from monomeric JC-1 increases approximately linearly with membrane (M) potential in the range of 46-182 mV, whereas 590 nm J-aggregate emission is less sensitive to M values below 140 mV and strongly sensitive to potential values in the range of 140-182 mV (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, respectively. Alternatively, a standard fluorescein long-pass optical filter set can be used to observe both forms simultaneously.

[0111] Dihydrorhodamine 123 is an uncharged, non-fluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123).

[0112] Measurement of the amount of mitochondrial outer membrane permeabilization from the test and reference populations can be performed in parallel. The sequential order of the steps is not required.

[0113] Alternatively, step (c) comprises or consists solely of measuring caspase activation.

[0114] Caspases (cysteine-aspartic acid proteases) are involved in the early stages of apoptosis. Caspase activation represents a key event in the early apoptotic process. The molecular events leading to caspase activation have been utilized in cell-free systems to detect apoptotic cells generated in the mitochondrial death pathway. Caspase activation can be detected by in vitro enzyme assays. Western blot mixtures are designed to detect proteins synthesized in response to apoptotic stimuli, such as caspases.

[0115] Quantitative reverse transcription PCR (RT-qPCR) is used when the starting material is RNA. 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, and PARP are recommended. In addition to early detection of apoptosis by Western blot analysis, caspase activity can be detected by in vivo caspase substrate cleavage, flow cytometry, fluorescence and light microscopy, and fluorescent ELISA. Fluorescent substrates or antibodies preferentially targeting caspase 3 and caspase 7 generate fluorescence 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 apoptosis in epithelial cells. Another antibody, anti-PARP, may be used to detect PARP fragments and intact PARP in apoptotic and healthy cell extracts by Western blot. Caspase cleavage of PARP can be measured by Western blot, immunohistology, and immunoprecipitation, as well as p53, annexin V, and M30 proteins.

[0116] Alternatively, step (c) can be carried out by measuring extracellular phosphatidylserine.

[0117] Annexin A5 or annexin V is a calcium-dependent phospholipid-binding protein that specifically binds to phosphatidylserine (PS). In healthy cells, PS is located exclusively on the cytosolic side of the plasma membrane. During the early stages of apoptosis, the asymmetric distribution of PS is lost and PS is translocated to the extracellular leaflet of the membrane. The presence of PS on the extracellular side of the plasma membrane can be detected using fluorescently labeled annexin V. Suitable dyes are impermeable dyes such as propidium iodide (PI), trypan blue, and 7-amino-actinomycin (7-AAD).

[0118] The amount of mitochondrial cell apoptotic death obtained from the test cell population is compared to the amount obtained from a reference cell population not contacted with the therapeutic agent.

[0119] Any statistical method may be used to control the measurements obtained.

[0120] The reference cell population can be a test cell population isolated from the same subject but not contacted with a therapeutic agent. Alternatively, the reference cell population is a cell population of the same nature as the test cell but derived from a healthy subject. By the expression "same nature", the present invention refers to the sample being isolated from the same organ / nature of the sample.

[0121] In one embodiment of the method of the third aspect of the invention, a kit as defined in the second aspect of the invention is used, alone or in combination with any of the embodiments provided above.

[0122] In another embodiment, the method of the third aspect of the invention comprises the use of a microfluidic device as defined above. In this embodiment, step (a) can be performed either inside or outside the microfluidic device. In one embodiment, step (a) is performed outside the microfluidic device.

[0123] Following this method, step (b) is performed in a microfluidic device and includes filling wells with the obtained cells and perfusing one or more BCL-2 anti-apoptotic family inhibitors in a buffer solution. 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 creating a concentration gradient. In another embodiment, optionally in combination with any of the embodiments provided above or below, the concentration gradient is created by perfusing culture medium alone through one inlet of the device and culture medium plus one or more BCL-2 inhibitors through a second inlet of the device.

[0124] Steps (c) to (e) are carried out as described in detail above.

[0125] In a further 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 described in detail above. [Example]

[0126] Cell culture and treatments GIST-T1 and GIST-T1 / 670 were kindly provided by Dr. Cesar Serrano at the Vall d'Hebron Institute of Oncology. These cells were cultured at 37°C and 5% CO2 in IMDM medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS) (10270106, ThermoFisher Scientific), 1% penicillin / streptomycin (15140122, Gibco ThermoFisher Scientific), and 1% L-glutamine (25030024, ThermoFisher Scientific).

[0127] The NALM-6 and SEM cell lines were kindly provided by the laboratory of Professor Pablo Menendez at the Josep Carreras Leukemia Research 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 maintained in a humidified incubator at 37°C and 5% CO2.

[0128] Imatinib and gefitinib were obtained from LC Laboratories (Woubourn, MA, USA), trametinib from SelleckChem (Munich, Germany), and ABT-199, A-1331852, and S6385 from MedChemExpress (Monmouth Junction, NJ, USA).

[0129] A 1 mM BH3 mimetic combination was prepared by adding 0.1 mM ABT-199, 0.1 mM A-1331852 and 1 mM S63845.

[0130] Dynamic BH3 profiling by flow cytometry (for comparison) BCP-ALL and GIST cells were pretreated with different anticancer drugs for 16 hours, stained with Zombie Violet to mark viable cells, washed with bovine phosphate buffered saline (PBS), and resuspended in mannitol experimental 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, 0.03, 0.1, 0.3, 1, 3, and 10 μM BIM peptide, 25 μM alamethicin, and DMSO-only control. After 1 hour of incubation at room temperature, cells were fixed with 8% formaldehyde, neutralized with N2 buffer (1.7 M Tris base, pH 9.1, 1.25 M glycine), and stained with intracellular staining buffer (1% Tween 20, 5% BSA in PBS) containing 1:1000 cytochrome c antibody conjugated to Alexa Fluor 647®. After overnight incubation at 4°C, results were acquired on an LSRII flow cytometer and processed using FlowJo software according to the manufacturer's instructions. A gate for % cytochrome c retained was created for the untreated condition and applied to all conditions.

[0131] Real-time tracking of apoptosis (RTA) by flow cytometry (present invention) Cells were seeded in 12-well plates and treated with different anticancer drugs for 16 hours. After incubation, cells were trypsinized, stained with Zombie Violet, and washed with PBS. Cells were then resuspended in the appropriate complete medium, and 25 μL of each cell solution was added to a 96-well plate containing 25 μL of complete medium and different dilutions of various apoptosis-catalyzing agents: Combination A: 1x ABT-199, 1x A1331852, and 10x S63845; Combination B: 1x S55746, 1x WEHI-539, and 10x AZD5991; and Combination C: 1x ABT-199, 1x DT2216, and 10x AZD5991. For 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 were incubated at 37°C for 1 hour for BCP-ALL cell lines and 3 hours for GIST cell lines, followed by fixation with 8% PFA, neutralization with N2 buffer, and staining using an antibody against cytochrome c. Because BCP-ALL cells are nonadherent, after overnight incubation at 4°C, results were acquired using an LSRII flow cytometer and processed using FlowJo software. A gate for the % retained cytochrome c was created for the untreated condition and applied to all conditions. The same protocol was also performed, but replacing the complete medium with FBS-free medium.

[0132] RTA by fluorescence microscopy (present invention) 100,000 cells were resuspended in 1 mL of complete medium and treated with the corresponding anticancer drug. 100 μL of the cell solution was seeded into eight wells of a 96-well plate for each treatment condition. Cells were incubated for 16 hours. After incubation, cells were stained with 2 μM calcein AM and 100 nM TMRE in complete medium (the medium used for cell culture) for 30 minutes at 37°C, washed with PBS, and exposed to the apoptosis catalyst combination of the present invention under the same conditions as in the flow cytometry version. Plates were incubated at 37°C for 3 hours and images were captured with a Nikon Eclipse Ti microscope. CellProfiler was used to automatically identify cells using the calcein AM field, quantify TMRE intensity, and distinguish apoptotic cells from non-apoptotic cells by applying a threshold of the 90% percentile of the fluorescence intensity in the control condition.

[0133] SU8 type manufacturing To develop the pattern, a silicon wafer (4" n-type <100> The wafer (MicroChemicals GmbH, Ulm, Germany) was cleaned in a PCD-002-CE Plasma Cleaner (Harrick Plasma, Ithaca, NY, USA) at 6.8 W for 20 minutes and then heated on a hotplate at 95 °C for 5 minutes. Next, SU-8 photoresist (2100, MicroChem Lab, Westboro, MA, USA) was spin-coated onto the wafer (first at 500 rpm with an acceleration of 100 rpm / s for 5 seconds, followed by 3000 rpm with an acceleration of 401 rpm / s for 30 seconds) to obtain a 100 μm-thick layer. The wafer was soft-baked at 65 °C for 5 minutes and 95 °C for 20 minutes for solvent evaporation. A negative photoresist mask printed on high-quality acetate film was used to pattern the microfluidic chip design using 240 mJ / cm2 energy radiation. The photoresist was crosslinked by exposure on a hotplate at 65 °C for 5 minutes and 95 °C for 10 minutes. The unstable photoresist was then removed by immersion in SU-8 developer (Y020100, MicroChem Lab) for 10 min and washed with 2-propanol. Finally, the wafer was placed on a hotplate at 150 °C for 60 min and finally cooled down to room temperature, where it was silanized to obtain a hydrophobic surface.

[0134] Microfluidic chip fabrication To obtain the microfluidic chip, PDMS was prepared by mixing Sylgard 184 (Dow Corning, Midland, Michigan, USA) prepolymer with a curing agent in a 1:10 ratio, followed by degassing in a cavity chamber for 1 hour. Three different layers were prepared, and the polymer volume was calculated to obtain the desired thickness of the PDMS layer. For the first 2 mm-thick layer, the PDMS prepolymer was poured onto an SU8 master mold with the design motif fixed inside a plastic Petri dish. For the second 1 mm-thick layer, the prepolymer was dispensed directly into an empty Petri dish. For the third layer, a clean 75 x 50 mm glass slide (CLS294775X50, Sigma-Aldrich) was pressed onto the uncured polymer mix to obtain a thin layer of PDMS on the glass. The PDMS was placed on a flat surface at room temperature and then heated to 85 °C for 4 hours. The PDMS layer was then carefully peeled off, and holes for the inlets and outlets were punched into the 2 mm layer using a 0.5 mm biopsy punch. Both layers (2 mm and 1 mm thick) were irreversibly bonded by plasma activation, baked at 85 °C for 4 h, and punched out using a 4 mm biopsy punch to create the cell chamber well. The resulting 3 mm thick PDMS layer was irreversibly bonded to a thin PDMS layer on a glass slide and heated at 85 °C for 4 h. Finally, a second glass slide was used to cover the well during the experiment.

[0135] Microfluidic RTA (present invention) The chip was placed in an oven at 85°C for 1 hour. 10 μL of sterile MiliQ water containing 15 μg / mL poly-L-lysine was added to each well and incubated at 37°C for 40 minutes to coat the well surface, followed by a washing step with MiliQ water. 100,000 cells were resuspended in 600 μL of complete medium (used for cell culture) and separated into three separate tubes containing the appropriate treatment concentrations. 35 μL of the cell suspension was added to each well, and the chip was incubated at 37°C for 16 hours. After incubation, the wells were refilled with complete medium and sealed with a glass slide secured with plastic alligator clips. Complete medium containing 100 nM TMRE and 2 μM calcein AM was then perfused through the two inlets at 300 mbar pressure using a P2SC pump for 15 minutes, and the chip was incubated at 37°C for 40 minutes. Complete medium was then perfused through one inlet at 200 mbar for 20 minutes, while the other inlet was perfused with the same buffer plus 1 μM of a combination of apoptosis-catalyzing agents of the present invention. The chip was incubated at room temperature for 2 hours, after which images were acquired using a Nikon fluorescence microscope.

[0136] Gradient characterization A solution containing 10 mg / mL bovine serum albumin (BSA) (A3059, Sigma-Aldrich) in MiliQ water and 428 μL / mL blue food dye (Vahine, Catalonia, Spain) was perfused through the first inlet and MiliQ water alone through the second inlet at 200 mbar using a precision pressure control system P2CS pump (Biophysical tools, Leipzig, Germany). The liquid exiting the outlet was collected in an Eppendorf tube. The BSA concentration was measured using a Pierce™ BCA Protein Quantitation Kit (23225, ThermoFisher Scientific), and the absorbance at 640 nm blue wavelength was measured using a Benchmark Plus Microplate Reader (4100172C, Bio-Rad, California, USA). Fluorescence gradient characterization was performed by injecting 25 μg / mL fluorescein (F2456, Sigma-Aldrich) in 10 mM NaOH and capturing images with a 436 ZEISS Axio Observer Z1 / 7 microscope.

[0137] statistical analysis All results were expressed as the mean ± SEM of at least three biologically independent replicate experiments. All conditions were compared to the control condition using an unpaired t-test, and p values less than 0.05 were marked as statistically significant (*). Statistical analysis was performed and results were presented using GraphPad Prism 9.

[0138] result Real-time tracking of apoptosis identifies effective treatments in liquid tumors The inventors compared the efficiency of apoptosis priming using the combination of the present invention with the methodology already reported in the prior art WO2014047342 (hereinafter also referred to as "standard DBP").

[0139] In the NALM-6 cell line, RTA exhibited a dose-response curve similar to that of standard DBP, demonstrating that titration of the combination of the apoptosis-catalyzing agent of the present invention had similar effects to the BIM peptide. Importantly, in cells treated with trametinib, lower concentrations of both the apoptosis-catalyzing agent and the BIM peptide were required to initiate the apoptotic process, whereas in the case of imatinib, the same curve was obtained as in the control condition (Figure 1A and C).

[0140] Similar results were obtained in SEM cells, where SEM cells treated with sunitinib were more sensitive to the apoptosis catalytic agent and BIM peptide of the present invention and showed increased apoptotic priming that correlated with future cytotoxicity (Figures 1B and 1D).

[0141] The results of these types of assays can also be expressed as Δ% priming, which represents the difference in apoptotic priming between the treatment and control conditions at a given concentration in the titration. Comparing DBP and RTA results, an increase in Δ% priming was obtained after treatment with trametinib in NALM-6 and sunitinib in SEM, but no increase in apoptotic priming was observed in either cell line after imatinib (Figures 1E and 1F). These results demonstrate that the new RTA protocol can identify effective treatments in BCP-ALL cell lines with efficacy equal to or greater than that of standard DBP, with the notable advantage of not requiring cells to be permeabilized with digitonin.

[0142] In solid tumors, RTA can be used in conjunction with flow cytometry and fluorescence microscopy To demonstrate that the new assay also works in adherent cells, we used two gastrointestinal stromal tumor (GIST) cell lines, GIST-T1 and GIST-T1 / 670. As previously published, the GIST-T1 cell line is sensitive to imatinib treatment, whereas GIST-T1 / 670 has acquired resistance to this inhibitor.

[0143] Again, standard DBP was performed on GIST-T1 cells after treatment with imatinib and, as a negative control, gefitinib. Pretreatment with imatinib reduced the concentration of BIM peptide required to initiate the apoptotic process compared with the untreated condition, whereas treatment with gefitinib showed a similar profile to the control (Figure 2A). In the case of the GIST-T1 / 670 cell line, the three experimental conditions had the same dose-response curve, as neither imatinib nor gefitinib was effective in these cells (Figure 2B).

[0144] RTA was performed using the apoptosis catalyst of the present invention, and a dose-response curve was obtained when flow cytometry was used for reading. Similar to the results in BCP-ALL, treatment with imatinib in GIST-T1 caused a decrease in the concentration of apoptosis inducers, initiating the process, indicating an increase in apoptosis priming after treatment with effective drugs (Fig. 2C). Again, treatment with imatinib in the resistant cell line GIST-T1 / 670 or gefitinib in both cell lines resulted in a curve identical to the control condition (Fig. 2D).

[0145] The new protocol provided by this invention avoids cell permeabilization with digitonin but fixes cells at the end of the incubation to allow immunostaining for cytochrome C. To improve upon this, a new methodology was developed based on TMRE, a fluorescent dye that accumulates in healthy mitochondria (non-apoptotic cells) but loses fluorescence when mitochondria are permeabilized (apoptotic cells). To demonstrate that this microscope version achieves similar results, RTA was adapted to incorporate this dye. GIST-T1 cells were found to provide a dose-response curve based on the intensity of TMRE, which differentiated apoptotic from non-apoptotic cells. Importantly, pretreatment with imatinib caused a shift in the curve, identifying increased apoptotic priming, whereas this was not observed in gefitinib-treated cells (negative control) (Figure 2E). As expected, in the case of GIST-T1 / 670 cells, both treatments resulted in an unchanged curve, similar to the control condition (Figure 2F).

[0146] When Δ% priming was quantified for all three methods, similar results were obtained in all cases, with a clear increase in apoptotic priming after imatinib treatment in GIST-T1 cells and no significant changes in other conditions (Figures 2G and 2H). This demonstrates that RTA can be performed in adherent cells using both flow cytometry and fluorescence microscopy, avoiding, in the latter case, the immunostaining step using TMRE as a fluorescent marker of apoptosis induction.

[0147] RTA works in conjunction with other apoptosis catalytic agents of the present invention The first experiment focused on the apoptosis catalytic agent created by ABT-199, A1331852 and S63845, designated Combination A.

[0148] To demonstrate that RTA can be performed using any combination that simultaneously affects BCL-2, BCL-xL, and MCL-1, two new apoptosis catalytic agents were prepared.

[0149] Combination B was made using three different direct inhibitors of the aforementioned anti-apoptotic proteins (S55746, WEHI-539 and AZD5991).

[0150] Combination C was created by combining the direct inhibitors of the other combinations (ABT-199 and AZD5991) with the addition of DT2216, a PROTAC drug that affects BCL-xL by its degradation, acting as an indirect inhibitor.

[0151] The same experimental setup was first performed with the BCP-ALL cell lines NALM-6 and SEM.

[0152] Cytometric RTA using a combination of B and C in the NALM-6 cell line achieved a dose-response curve in the untreated and imatinib-treated conditions, but in the trametinib-treated condition, less apoptosis-catalyzing agent was required to induce apoptosis in both cases (Figures 3A and 3C).

[0153] In SEM cells, results were similar, showing induction of apoptotic priming after treatment with sunitinib in both combinations B and C when compared to untreated cells (Figures 3B and 3D).

[0154] Finally, a comparison of Δ% priming between standard DBP and RTA using three combinations of apoptosis-catalyzing agents yielded similar results between the methods.

[0155] In all cases, trametinib in NALM-6 and sunitinib in SEM caused and increased apoptotic priming (Figures 3E and 3F).

[0156] Using models of adherent cells, GIST-T1 and GIST-T1 / 670, RTA was performed after anticancer treatment using novel combinations of apoptosis-catalyzing agents.

[0157] 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 (Figures 4A and 4B).

[0158] In the case of Combination C, untreated cells did not respond to increasing concentrations of the apoptosis-catalyzing agent, likely due to the mechanism of effect and incubation time of DT2216. Nevertheless, in imatinib-treated GIST-T1 cells, there was a shift in the dose-response curve caused by Combination C, indicating the induction of apoptotic priming and the identification of an effective drug (Figures 4C and 4D). Again, when comparing the Δ% priming caused by the three inventive combinations and standard DBP, imatinib in GIST-T1 cells was the only condition that showed an increase in apoptotic priming (Figures 4E and 4F).

[0159] These results demonstrate that apoptosis induction of anti-cancer treatments can be specified using any combination acting on three major anti-apoptotic proteins (BCL-2, BCL-xL, and MCL-1), both by direct inhibition or indirect targeting of the proteins.

[0160] RTA can be performed in a microfluidic chip One of the major limitations of both classical DBP and early RTA experiments is that they both required large numbers of viable cells to perform the assay. We have incorporated this combination into a microfluidic chip, which significantly reduces the number of cells required and allows for the automation of the process, aiding its implementation in the clinic.

[0161] GIST-T1 cells were seeded and treated within a microfluidic device, and a concentration gradient of BIM peptide was exposed to the cells directly generated by the device. Increasing concentrations of BIM peptide induced the initiation of apoptosis in control cells, as shown by the resulting dose-response curve. The same cells treated with imatinib required lower concentrations of peptide, demonstrating increased apoptotic priming (Figure 5A).

[0162] As previously demonstrated, RTA using Combination A can identify the induction of apoptotic priming in GIST-T1 cells after treatment with imatinib using fluorescence microscopy (Figure 5B). Using the same protocol, we seeded GIST-T1 cells in a microfluidic device and treated them with imatinib and gefitinib (negative control). A titration of the apoptotic catalyst was performed in the device itself, and a dose-response curve was generated using TMRE to identify apoptotic and non-apoptotic cells. Untreated GIST-T1 cells responded identically to Combination A as gefitinib-treated cells, but less apoptotic catalyst was required to initiate the process in imatinib-treated cells (Figure 5C). When quantification of Δ% priming was performed, the microfluidic version of RTA, similar to the microfluidic versions of DBP and microscope-based RTA, was able to identify increased apoptotic priming caused by imatinib in GIST-T1 cells (Figure 5D).

[0163] The microfluidic device could identify effective treatments using a combination of BH3 mimetics as the apoptosis catalytic agent combination 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 anti-cancer drugs in patient samples in situ in the hospital.

[0164] Terms For completeness, various aspects of the invention are set forth in the following numbered clauses. Clause 1. A combination comprising a BCL-2 inhibitor, a BCL-xL inhibitor and an MCL-1 inhibitor. Clause 2. The combination of clause 1 consisting solely of a BCL-2 inhibitor, a BCL-xL inhibitor and an MCL-1 inhibitor. Clause 3. Any combination of any one of the preceding clauses, including or consisting only of ABT-199, A-1331852 and S63845. Clause 4. A kit comprising a combination of inhibitors as defined in any one of clauses 1 to 3. Clause 5. The kit of clause 4, which is a microfluidic device. Clause 6. A kit according to clause 5, comprising means for generating a gradient, in particular a logarithmic gradient, within said device. Clause 7. A kit according to any one of clauses 6 to 7, including a T-junction gradient generator. Clause 8. The combination of any one of clauses 1 to 3 or the kit of any one of clauses 4 to 7, wherein said combination further comprises one or more additional anti-apoptotic BCL-2 family inhibitors having a molecular weight of less than 2KDa, particularly less than 1.5KDa, in particular less than 1KDa. Clause 9. A method for predicting the cytotoxicity of a therapeutic agent to a cell, said method comprising: a) contacting an isolated test cell population with a test therapeutic agent; b) contacting the cell population resulting from step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein said one or more anti-apoptotic BCL-2 family inhibitors have a molecular weight of less than 2 KDa, particularly less than 1.5 KDa, in particular less than 1 KDa; c) determining the apoptosis priming value; d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from said subject with said one or more anti-apoptotic BCL-2 family inhibitors used in step (b) above; e) comparing both priming values; - step (d) is carried out in the absence of said therapeutic agent; - the process is carried out in the absence of a cell permeabilizing agent, wherein a decrease in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent is cytotoxic to the cells. Clause 10. A method for predicting cytoprotection of a therapeutic agent, said method comprising: a) contacting an isolated test cell population with a test therapeutic agent; b) contacting the cell population resulting from step (a) with one or more anti-apoptotic BCL-2 family inhibitors, wherein said one or more anti-apoptotic BCL-2 family inhibitors have a molecular weight of less than 2 KDa, particularly less than 1.5 KDa, in particular less than 1 KDa; c) determining the apoptosis priming value; d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from said subject with said one or more anti-apoptotic BCL-2 family inhibitors used in step (b) above; e) comparing both priming values; - step (d) is carried out in the absence of said therapeutic agent; - the process is carried out in the absence of a cell permeabilizing agent, wherein an increase in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent protects the cells. Clause 11. The method of any one of clauses 9 to 10, wherein the priming values are determined at different concentrations of the one or more anti-apoptotic BCL-2 family inhibitors, thus obtaining a priming curve for each of the test sample and the reference sample. Clause 12. If the priming curve of the test sample is shifted to the left relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent is cytotoxic to the cells; 12. The method of clause 11, wherein if the priming curve of the test sample is shifted to the right relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent protects the cells. Clause 13. The method of any one of clauses 9-12, wherein steps (c) and (d) are carried out by measuring mitochondrial outer membrane permeabilization in said test cell population and said reference cell population. Clause 14. The method of any one of clauses 9 to 13, wherein measuring said mitochondrial outer membrane permeabilization comprises measuring the release of molecules from the mitochondrial intermembrane space, and in particular comprises the release of one or more of the following molecules from said mitochondrial intermembrane space: cytochrome c, SMAC / Diablo, Omi, adenylate kinase-2 or apoptosis-inducing factor (AIF), and in particular comprises the release of cytochrome c. Clause 15. The method of any one of clauses 9 to 14, wherein said cell population is a cancer cell population and said therapeutic agent is a chemotherapeutic agent, particularly a targeted chemotherapeutic agent, particularly a kinase inhibitor.

Claims

1. In vitro non-therapeutic use of a combination comprising a BCL-2 inhibitor, a BCL-xL inhibitor and an MCL-1 inhibitor to prime an apoptotic state in an isolated test cell sample.

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

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

4. The in vitro use according to any one of claims 1 to 3, wherein the combination comprises a direct inhibitor of BCL-2 and MCL-1 and an indirect inhibitor of BCL-xL.

5. 5. The in vitro use according to any one of claims 1 to 4, wherein the combination comprises or consists of one of: ABT-199, A-1331852 and S63845; ABT-199, DT2216 and AZD5991; and S55746, WEHI-539 and AZD5991.

6. A kit comprising a combination of inhibitors as defined in any one of claims 1 to 5.

7. The kit of claim 6, which is a microfluidic device.

8. 8. The 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. 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 further anti-apoptotic BCL-2 family inhibitors having a molecular weight of less than 2 KDa, in particular less than 1.5 KDa, in particular less than 1 KDa.

11. 1. A method for predicting a subject's response to a treatment, said method comprising: (a) contacting a test cell sample isolated from said subject with a candidate drug; (b) priming the apoptotic state by contacting the cell sample with a combination as defined in any one of claims 1 to 5; (c) determining a priming value for the test sample; (d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from said subject with one or more anti-apoptotic BCL-2 family inhibitors used in the previous step (b); (e) comparing both priming values; A method comprising:

12. 1. A method for predicting the cytotoxicity of a therapeutic agent to a cell, said method comprising: a) contacting an isolated test cell population with a test therapeutic agent; b) priming an apoptotic state of the cells by contacting the cell population resulting from 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, particularly less than 1.5 KDa, and particularly less than 1 KDa; c) determining the apoptosis priming value; d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from said subject with said one or more anti-apoptotic BCL-2 family inhibitors used in step (b) above; e) comparing both priming values; - step (d) is carried out in the absence of said therapeutic agent; - the process is carried out in the absence of a cell permeabilizing agent, wherein a decrease in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent is cytotoxic to the cells.

13. 1. A method for predicting cytoprotection of a therapeutic agent, said method comprising: a) contacting an isolated test cell population with a test therapeutic agent; b) priming an apoptotic state of the cells by contacting the cell population resulting from 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, particularly less than 1.5 KDa, and particularly less than 1 KDa; c) determining the apoptosis priming value; d) obtaining a reference apoptotic priming value by contacting another isolated cell population sample from said subject with said one or more anti-apoptotic BCL-2 family inhibitors used in step (b) above; e) comparing both priming values; - step (d) is carried out in the absence of said therapeutic agent; - the process is carried out in the absence of a cell permeabilizing agent, wherein an increase in the priming value from the test cell portion compared to the priming value of the reference cell portion indicates that the therapeutic agent protects the cells.

14. 14. The method of any one of claims 11 to 13, wherein the priming values are determined at different increasing concentrations of the one or more anti-apoptotic BCL-2 family inhibitors, thus obtaining a priming curve for each of the test sample and the reference sample.

15. if the priming curve of the test sample is shifted to the left relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent is cytotoxic to the cells; 15. The method of claim 14, wherein if the priming curve of the test sample is shifted to the right relative to the priming curve of the reference sample, this indicates that the tested therapeutic agent protects the cells.

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

17. 17. The method of any one of claims 11 to 16, wherein measuring mitochondrial outer membrane permeabilization comprises measuring the release of molecules from the mitochondrial intermembrane space, in particular 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 of any one of claims 11 to 17, wherein the cell population is a cancer cell population and the therapeutic agent is a chemotherapeutic agent, particularly a targeted chemotherapeutic agent, in particular a kinase inhibitor.

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

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