Tubulin acetylation and its associated post-translational modifications as biomarkers of response to treatment with taxol, taxanes and other microtubule stabilizing Anti-cancer agents

Abstract

The present invention discloses high α-tubulin acetylation as a biomarker for cancer cell response to taxol. In one embodiment the present invention refers to a method for predicting prognosis of treatment with taxol, taxanes and other microtubule (MT) stabilizing anti-cancer agents, comprising a step of assessing the level of α-tubulin acetylation in cancer cells. The present invention further refers to a composition for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents by assessing the presence or amount of α-tubulin acetylation in cancer cells, the said composition comprising a reagent to detect acetylated α- tubulin, a non-limiting example of which comprises anti-acetylated α-tubulin (acetyl K40) antibody. The present invention further refers to a kit for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents, the kit comprising the above- mentioned composition and the means for determining the presence or amount of α-tubulin acetylation in cancer cells, including controls. In another embodiment, the present invention refers to method, compositions and kit for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti- cancer agents for a patient, comprising the steps of assessing the presence or amount of α-tubulin acetylation in cancer cells and; b) adjusting the treatment regimen in regards to dose, infusion time, number of treatment cycles, duration of treatment cycles, duration of intervals between treatment cycles and overall duration of treatment, and combinations thereof. The present invention's methods, compositions and kits can be advantageously used for cancer patient stratification towards a more personalized treatment with MT-targeting drugs.

Description

[0001] DESCRIPTION

[0002] TUBULIN ACETYLATION AND ITS ASSOCIATED POST-TRANSLATIONAL MODIFICATIONS AS BIOMARKERS OF RESPONSE TO TREATMENT WITH TAXOL , TAXANES AND OTHER MICROTUBULE STABILIZING ANTI-CANCER AGENTS

[0003] Technical field of the invention

[0004] The present invention relates to the technical field of physics , testing through investigating or analysing materials by determining the chemical or physical properties of biological material , the said testing involving immunoassay, biospeci fic binding assay, and materials therefore , for cancer testing involving markers for tumor, cancer or neoplasia ( G01N33 / 57484 ) .

[0005] Prior art

[0006] Microtubules (MTs ) are dynamic polymers of a / p-tubulin involved in cell division, migration and invasion, and are amongst the most success ful targets in cancer treatment ( Dumontet and Jordan, 2010 ) . Human cells express several a / p-tubulin isotypes that combine with various post-translational modi fications ( PTMs ) to generate MT diversity ( Janke and Magiera, 2020 ; Verhey and Gaertig, 2007 ) . How this so-called "tubulin code" is read by MT-associated proteins (MAPs ) and motors and whether it impacts speci fic cellular functions remained largely unknown for over 40 years . However, with the discovery of the catalytic enzymes accounting for the di f ferent tubulin PTMs , recent studies started to unveil their critical roles in mitosis and meiosis , neuronal processes and brain function, as well as heart and s keletal muscle contraction (Magiera et al . , 2018 ) . Importantly, despite existing evidence linking speci fic tubulin isotypes and PTMs with tumor development and metastasis , a comprehensive analysis and dissection of the "cancer tubulin code" is still lacking ( Lopes and Maiato , 2020 ) . a-tubulin acetylation of Lysine 40 (K40 ) and detyrosination are amongst the best characteri zed tubulin PTMs ( Janke and Magiera, 2020 ; Verhey and Gaertig, 2007) . K40 acetylation, which takes place in the MT lumen, is mediated by aTATl (Akella et al., 2010; Shida et al., 2010) and reverted by HDAC6 and SIRT2 deacetylases (Hubbert et al., 2002; North et al., 2003) . In turn, detyrosination consists in the catalytic removal of the last tyrosine residue from the unstructured C-terminal tail (CTT) of most a-tubulin isotypes. This is mediated in part by VASH1 / VASH2 carboxypeptidases associated with the small activating peptide SVBP (Aillaud et al., 2017; Nieuwenhuis et al., 2017) , and reverted by a highly specific tubulin tyrosine ligase (TTL) that re-tyrosinates a-tubulin (Ersfeld et al., 1993) . A non-re-tyrosinatable A2-a-tubulin may also form after the removal of the penultimate glutamic acid in the CTT by cytosolic carboxypeptidases ( Paturle-Laf anechere et al., 1991) . Acetylated, detyrosinated and A2-a-tubulin accumulate on stable / long-lived MTs (Khawaja et al., 1988; Paturle- Lafanechere et al., 1994; Webster et al., 1990) . As so, cell treatment with MT stabilizing drugs, such as taxol / paclitaxel , increases the accumulation of these tubulin PTMs (Ferreira et al., 2020; Paturle-Laf anechere et al., 1994; Webster et al., 1990; Xiao et al., 2006) , but their functional relationship and whether they directly or indirectly interfere with MT dynamic properties remains unclear. Recent works suggested that a-tubulin acetylation of K40 confers resistance to MT breakage, thereby promoting the mechanical stability of long-lived MTs (Fortran et al., 2017; Xu et al., 2017) . In contrast, MT depolymerization experiments and injection of function-blocking antibodies against TTL suggested that a-tubulin detyrosination does not directly interfere with MT stability (Khawaja et al., 1988; Webster et al., 1990) . More recently, genetic perturbation of TTL in human cells and in vitro reconstitution experiments further supported this conclusion (Chen et al., 2021; Ferreira et al., 2020) . However, a-tubulin detyrosination suppresses the activity of effector proteins involved in the regulation of MT dynamics, including MCAK, a MT- depolymerizing enzyme of the kinesin-13 family (Ferreira et al., 2020; Liao et al., 2019a; Peris et al., 2009; Sirajuddin et al., 2014) . Intrinsic or acquired resistance to MT-targeting drugs, such as taxol and its derivatives, remains a major challenge in improving therapy response and cancer patient survival (Orr et al., 2003) . Several mechanisms, including altered expression of tubulin isotypes, tubulin mutations and modifications of MT- regulatory proteins, have been implicated in taxol resistance, but whether and how tubulin PTMs play a role in response to taxol remains a problem.

[0007] Definitions

[0008] Taxanes : In the present disclosure, taxanes mean paclitaxel, docetaxel, cabacitaxel, albumin bound paclitaxel (abraxene) , as well as any other taxanes or taxol derivatives.

[0009] Tubulin acetylation: In the present disclosure tubulin acetylation further means other tubulin post-translational modifications which correlate with tubulin acetylation.

[0010] Summary of the Invention

[0011] Using the NCI-60 cancer cell panel to perform a comprehensive analysis and initial dissection of the cancer tubulin code, the present invention discloses the respective impact in taxol-induced cytotoxicity .

[0012] In particular, the present invention discloses how specific post- translational signatures, namely a-tubulin acetylation, detyrosination and related delta-2 (A2) modification, vary among the different cancer cell lines of the panel.

[0013] The present invention reveals that elevated a-tubulin acetylation predicts taxol sensitivity and may be a useful biomarker for cancer patient stratification towards a more personalized treatment regimen with MT-targeting drugs.

[0014] Detailed description of the Invention The NCI-60 cancer cell panel a validated anticancer drug screening platform representing nine distinct tumour types (leukaemia, colon, lung, CNS, renal, melanoma, ovarian, breast and prostate) , Table 1.

[0015] Table 1. Summary of the NCI-60 cancer cell lines.

[0016] To assess whether distinct cancer cell types show specific molecular signatures associated with the accumulation of different a-tubulin PTMs, namely acetylation, detyrosination and related A2 modification, the NCI-60 cancer cell panel was screened by immunoblot with previously validated antibodies (Fig. 1A-C and Fig.2) . To control the quantitative immunoblot analyses, the detection of the re-tyrosinating enzyme TTL is included (Fig. ID and Fig. 2) and its expression is compared with the respective TTL mRNA levels obtained from the CellMinerTM database. A strong correlation (r=0.8272, p<0.0001) between TTL protein and mRNA levels among the different NCI-60 cell lines is found (Fig. 3A) , validating this approach. To compare different cancer cell lines between immunoblots, the leukemia cell line HL-60, which shows intermediate levels of a-tubulin acetylation and detyrosination, is included in each immunoblot as an internal reference (Fig. 2) . The human non-transformed hTERT-immortalized RPE1 cell line is also included in each immunoblot for qualitative comparative purposes (Fig. 2) . Subsequent quantification of the levels of the different tubulin PTMs and TTL reveals a high degree of variability among the different cancer cell lines, including within the same tissue, that in some cases varies more than 10-fold (Fig. 1A-D) . Importantly, these differences cannot be explained by cell cycle regulation of tubulin PTMs and TTL, since their respective levels do not change more than 1.5-fold throughout the cell cycle (Fig. 4) .

[0017] Because TTL accounts for a-tubulin re-tyrosination, high TTL expression is predicted to inversely correlate with a-tubulin detyrosination levels. Whether TTL protein expression correlates with a-tubulin detyrosination levels in the NCI-60 panel and likewise, whether a-tubulin detyrosination levels correlates with the related A2 modification and a-tubulin acetylation is herein disclosed. Analysis reveals that a-tubulin detyrosination levels only moderately correlate with A2-a-tubulin levels, with some cell lines, such as in SF-295 (CNS) , KM12 (colon) and PC-3 (prostate) , showing no obvious relationship between these PTMs (Fig. IB, C, Fig. 2B and Fig. 3) . In general, a-tubulin detyrosination shows a poor correlation with TTL protein expression (negative correlation) and with a-tubulin acetylation levels (positive correlation) among the different cancer cell lines of the NCI-60 panel (Fig. 3C, D) . Interestingly, a more detailed analysis focusing on the cell lines with higher a-tubulin detyrosination levels (>60th percentile) , revealed that 68.4% were found to express low TTL protein levels, 76.5% showed high A2-tubulin and 61.1% had high a-tubulin acetylation levels. In agreement, for the cell lines with lower (<40th percentile) a-tubulin detyrosination levels, 78.6% expressed high TTL protein levels, 76.5% had low A2- a-tubulin and 64.3% showed low a-tubulin acetylation levels (Fig. 3B' -D' ) .

[0018] In another embodiment of the present invention, association of specific tubulin PTMs with cancer cell cytotoxicity induced by treatment with the microtubule-stabilizing drug taxol is assessed. Assessment of a link between a-tubulin acetylation, detyrosination or A2-tubulin with taxol sensitivity reveals a weak, yet signi ficant , correlation between a-tubulin acetylation levels and taxol sensitivity among the NCI- 60 cancer cell panel ( Fig . 5A) . Surprisingly, focusing exclusively on the cancer cell lines with higher a-tubulin acetylation ( >60th percentile ) , a clear bias for improved taxol response is found ( Fig . 5A, A' ) .

[0019] In another embodiment of the present invention selected representative cell lines from the panel with either high (HCT- 116 , KM12 and MDA-MB-435 ) or low ( OVCAR-4 , UO-31 , HOP- 92 ) a-tubulin acetylation ( Fig . 5B, B' ) are employed to directly determine their response to taxol treatment . In l ine with the correlation analysis , these experiments show that cancer cells with high a-tubulin acetylation are more sensitive ( IC50 < 6 nM) to increasing concentrations of taxol , when compared with cancer cells with low a-tubulin acetylation that show increased taxol resistance ( IC50 > 24nM) ( Fig . 5C, O' ) . Altogether, the present invention reveals that high a-tubulin acetylation is a potential predictive biomarker for taxol cytotoxicity in cancer patients .

[0020] Altogether, regardless of any potential role of a-tubulin acetylation in taxol cytotoxicity on particular cancer types , a- tubulin acetylation represents a prognostic biomarker for taxol response .

[0021] In other embodiments , assessment of tubulin acetylation may comprise assays selected from the list consisting of : Immunohistochemistry, Immunocytochemistry, Immunoprecipitation, Mass-spectrometry and ELISA.

[0022] In another embodiment of the present invention, a method can be developed for predicting prognosis of treatment with taxol , taxanes or other microtubule (MT ) stabilizing anti-cancer agents , the said method comprising a step of assessing the level of a- tubulin acetylation in cancer cells . In another embodiment, the present invention refers to a composition for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents by assessing the presence or amount of a-tubulin acetylation in cancer cells in which the said composition comprises a reagent to detect acetylated a-tubulin, a non-limiting example of which comprises anti-acetylated a-tubulin (acetyl K40) antibody ab24610 from clone 6-11B-1.

[0023] In another embodiment of the present invention, a kit for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents can be developed, the said kit comprising means for determining the presence or amount of a-tubulin acetylation in a cancer cells, including controls .

[0024] In another embodiment of the present invention, a method can be developed for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents for a patient comprising the steps of: a) assessing the presence or amount of a-tubulin acetylation in cancer cells and; b) adjusting the treatment regimen in regard to dose, infusion time, number of treatment cycles, duration of treatment cycles, duration of intervals between treatment cycles, overall duration of treatment, and combinations thereof.

[0025] Regarding dose, regimen can be adjusted to 50mg / m2, 75mg / m2, 100 mg / m2, 125 mg / m2, 150 mg / m2, 175mg / m2, 200mg / m2, 225mg / m2, 250mg / m2, 275mg / m2or 300mg / m2.

[0026] Regarding infusion time, regimen can be adjusted to 1 hour (h) , 2h or 3h .

[0027] Regarding number of treatment cycles, regimen can be adjusted to 2, 4, 6, 8, 10, 12, 14 or 16. Regarding duration of treatment cycles, regimen can be adjusted to 7 days, 14 days, 21 days, 28 days, 35 days or 42 days.

[0028] Regarding duration of treatment intervals, the regimen can be adjusted to 0 days, 7 days or 14 days.

[0029] Regarding overall duration of treatment, regimen can be adjusted to 6 weeks, 12 weeks, 18 weeks, 24 weeks, 30 weeks, 36 weeks, 42 weeks or 48 weeks.

[0030] Another aspect of the present invention refers to a composition for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents for a patient through assessing the presence or amount of a-tubulin acetylation in cancer cells in which the said composition is characterized by, comprising a reagent to detect acetylated a- tubulin, a non-limiting example of which comprises anti-acetylated a-tubulin (acetyl K40) antibody ab24610 from clone 6-11B-1.

[0031] In another embodiment, the present invention refers to a kit for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents for a patient characterized by, comprising the composition described above and the means for determining the presence or amount of a-tubulin acetylation in cancer cells, including controls.

[0032] The present invention' s methods and kits can be advantageously used for cancer patient stratification towards a more personalized treatment with MT-targeting drugs.

[0033] Brief description of the Figures

[0034] Figure 1. Cancer cells display highly variable tubulin PTM signatures. (A-D) Quantification of tubulin PTMs (a-tubulin detyrosination, acetylation, A2-tubulin) and the re-tyrosinating enzyme TTL levels from the immunoblot screening in cell lines of the NCI-60 panel, normalized to the loading control p-tubulin (mean ± s.d.; 2-6 independent experiments with two replicates each) . To compare cell lines between immunoblots, the cell line HL-60 (dashed line ) was added in all experiments as a reference value.

[0035] The 53 cancer cell lines of the NCI-60 panel used in this study were authenticated by genotyping and grown in their corresponding medium (Table SI) at 37°C with 5% C02 (exception of SW-620, grown without C02 due to the Leibovitz's L-15 Medium) . COLO205, HCT-116, DLD-1, HCT-15 and HL-60 cells were obtained from Ipatimup' s Cell Lines Bank (University of Porto, Porto, Portugal) . All the other NCI-60 cell lines were kindly provided by Monica Bettencourt-Dias (Institute Gulbenkian de Ciencia, Oeiras, Portugal) (Marteil et al., 2018) . Parental HeLa (kindly provided by Y. Mimori-Kiyosue, RIKEN BDR, Kobe, Japan) , hTERT RPE-1 (ATCC®, CRL-4000) , U2OS (kindly provided by S. Geley, Innsbruck Medical University, Innsbruck, Austria) and PA-GFPa-tubulin / mCherry-a-tubulin U2OS (kindly provided by R. Medema, NKI, Amsterdam, the Netherlands) were grown at 37 °C with 5% CO2 in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% of FBS (Gibco, Thermo Fisher Scientific) .

[0036] For Immunoblot, cells are trypsinized and pelleted by centrifugation at 1200 rpm for 5 min (to rule out variability introduced by different cell densities among the NCI-60 panel, pellets from adherent cells were collected from cultures at equivalent near-conf luence states, ~ 90%) . Pellets were washed with PBS and resuspended in cold lysis buffer (50 mM Tris HC1 pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP40, 0.5% Triton™ X-100) , supplemented (freshly added) with protease inhibitors. After 30 min on ice, protein samples were snap-frozen in liquid nitrogen and centrifuged at 14000 rpm for 8 min at 4°C. Then, supernatants were collected and the protein concentration determined by Bradford assay. 50 pg of total protein per sample were denatured in Laemmli sample buffer at 95°C for 5 min, separated by 12% (v / v) SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane using an iBlot Gel Transfer System (Invitrogen, Thermo Fisher Scientific) or TransBlot® Turbo™ Transfer System (Bio-Rad) . Membranes were blocked with TBST (TBS with 0.05% Tween 20) containing 5% nonfat dry milk, for 1.5 h at room temperature (RT) . The following primary antibodies (diluted in TBST with 1 % nonfat dry milk) were used to incubate membranes overnight at 4°C: rabbit anti-detyrosinated a- tubulin (Polyclonal; Abeam, ab48389; 1:2000; the antibody used in the NCI-60 screen) , rabbit anti-detyrosinated a-tubulin (1:10000) (Liao et al., 2019b) , rabbit anti-TTL (Polyclonal; Proteintech, 13618-1-AP; 1:5000) , mouse anti-acetylated a-tubulin (acetyl K40) (Monoclonal; [6-11B-1] ; Abeam, ab24610; 1:1000) , rabbit anti-A2 a- tubulin (Polyclonal; Merck Millipore, AB3203; 1:1000) , mouse anti- p-tubulin (Monoclonal; Sigma-Aldrich, clone TUB 2.1, T 5201; 1:1000) , mouse anti-Cyclin Bl (V152) (Monoclonal; Cell Signaling Technology, 4135; 1:500) , mouse anti-GAPDH (Monoclonal; Proteintech, 60004-1-Ig; 1:40000) , rabbit anti-MCAK (Polyclonal; Abeam, ab71706; 1:500) , mouse anti-glutamylation tubulin (GT335) (Monoclonal; AdipoGen, AG-20B-0020-C100; 1:500) , rabbit anti- polyglutamylation (polyE) (Polyclonal; AdipoGen, AG-25B-0030- C050; IN105; 1:1000) . After TBST washes (3x for 10 min) , membranes were incubated with HRP-conj ugated secondary antibodies (Jackson ImmunoResearch; 1:5000) (diluted in TBST with 1 % nonfat dry milk) for 1 h at RT . Signal was detected with Clarity Western ECL Substrate in a ChemiDoc XRS+ System (Bio-Rad) . Quantification of protein levels were performed in Image Lab software.

[0037] All statistical analysis were performed in GraphPad Prism 9. To analyze normal (Gaussian) distribution, Shapiro-Wilk normality test is used.

[0038] Figure 2. Immunoblot screen of tubulin PTMs in the NCI-60 cancer cell panel. Protein lysates of 53 NCI-60 cancer cell lines and hTERT RPE-1 cells, immunoblotted (2-6 independent experiments) for a-tubulin detyrosination, A2 and acetylation, TTL and p-tubulin (as loading control) . Representative immunoblots of the screen, containing two replicates for each cancer cell line.

[0039] Figure 3. a-tubulin detyrosination, A2 and acetylation can be uncoupled in cancer cells. (A) Correlation between TTL protein (our screen) and mRNA (z scores; CellMinerTM database) levels in NCI-60 cancer cells. (B-D) Correlation between levels of a-tubulin detyrosination and A2-tubulin, TTL, and a-tubulin acetylation in NCI-60 cancer cells. (B'-D' ) Proportion of cancer cell lines of the panel with high and low A2-tubulin, TTL, and a-tubulin acetylation in the two groups with high and low a-tubulin detyrosination. From the tubulin PTM level values, the fourth and fifth quintile (>60th percentile) were defined as a 'high' group and the first and second quintile (<40th percentile) as a 'low' group. Spearman correlation coefficient (r) and p values are indicated in the graphs.

[0040] Figure 4. Tubulin PTMs only vary slightly throughout the cell cycle. (A) Protein lysates (HeLa cells) of different time-points after release from double thymidine synchronization (and asynchronous lysates) , immunoblotted for cyclin Bl, TTL, a-tubulin detyrosination, A2 and acetylation, p-tubulin and GAPDH was used as loading control. (B) Quantification of cyclin Bl, TTL, a-tubulin detyrosination and acetylation levels, normalized to asynchronous levels and loading control (mean ± s.d.; each dot represents an independent experiment (mean from 1-2 replicates) ; one-way ANOVA (relative to asynchronous) ; **, P < 0.01; ****, p < 0.0001; ns, not significant) .

[0041] For double thymidine cell synchronization at Gl / S phase, HeLa cells were grown in DMEM supplemented with 10% FBS (T25 flasks) until ~30% confluence. Freshly prepared thymidine (Sigma-Aldrich, T1895) (in PBS) was added to a final concentration of 2 mM. After 24 h blocking, cells were washed 3x with pre-warmed PBS and incubated with fresh medium for a 10 h release. Then, thymidine was added (final concentration of 2 mM) for another 24 h. For the final release, cells were washed 3x with pre-warmed PBS, incubated with fresh medium, and collected at different time points (0 h; 3 h; 7 h; 9 h; 13 h; 16 h) for western blot analysis. An asynchronous culture, without thymidine treatment, was used as a control. Average transcript intensity z scores of NCI-60 cell lines were obtained from the CellMiner database version 2.6 and 2.7 (https: / / discover.nci.nih.gov / cellminer / ) , using the analysis tool "Gene transcript level z score".

[0042] The nonparametric Spearman correlation is used in all correlations. Comparison between time-points were performed using one-way ANOVA. For the analysis, ****p < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant.

[0043] Figure 5. High a-tubulin acetylation correlates with taxol cytotoxicity. (A) Correlation between a-tubulin acetylation levels in the NCI-60 cancer cells and taxol activity z scores (Spearman correlation coefficient (r) and p value indicated) . (A' ) Average (and range) taxol activity z score between cell lines of the two groups high (>60th percentile) and low (<40th percentile) a- tubulin acetylation (Mann-Whitney test; *, P < 0.05) . (B, B' ) Representative Immunoblot and respective quantification (relative to p-tubulin) in the cell lines with high or low a-tubulin acetylation. (C) Graphic representation of cell viability after 120 h of taxol treatment (increasing concentrations) (Graphic representation of one experiment; lines show nonlinear curve fittings; error bars in each concentration show ± s.d. of 3-4 replicates) . (C' ) Related IC50 of 4 (HCT-116, KM12) and 3 (MDA- MB-435) independent experiments (mean ± s.d.; each dot represents an independent experiment) .

[0044] Taxol (NSC 125973) average activity z scores were obtained from the CellMiner database version 2.6, using the analysis tool "Drug activity level z score". For the taxol cytotoxicity experiments, taxol (Sigma-Aldrich, T7191) is serially diluted (24; 12; 6; 3; 1.5; 0.75; 0.375 nM) and the different concentrations added for 120 h. The same concentrations of taxol are added in all other cytotoxicity experiments for 72 h. Pharmacological inhibition of HDAC6, and increasing of a-tubulin acetylation is obtained using 1.5 pM of Tubastatin A (Selleck Chemicals, S8049) for 24 h. 5 pM of MG132 (Calbiochem, 474790) is used to induce metaphase arrest (less than 2 h of treatment to avoid cohesion fatigue) . For livecell and fixed cell analysis, 6 nM of taxol is added before acquisition or for 24 h before fixation, respectively. DMSO is used as control for these drugs. To assess cell viability, cells with different treatments are seeded in 96-well plates and in the following day treated with increasing concentrations of taxol (0- 24 nM; serially diluted) for 72h (Fig. 4B, D and Fig. 6C-E) or 120h (Fig. 3C) . Then, medium was removed and cells washed with PBS. Fresh medium containing 0.02 mg / mL of resazurin sodium salt (Sigma-Aldrich, R7017) was added and cells kept at 37°C in humidified conditions with 5% CO2, protected from light. After 4 h, supernatant medium was collected to a new 96-well plate and resorufin fluorescence monitored in a Synergy MX microplate reader using 530 nm excitation and 590 nm emission. Mann-Whitney test was used to analyze differences between average taxol activity z score in the groups with different a-tubulin acetylation levels. IC50 calculations were also performed in GraphPad Prism 9, using recommended instructions. Unpaired t test was used to analyze IC50 quantifications. Comparison between IC50 quantifications were performed using one-way ANOVA. For the analysis, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant.

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[0093] Lisbon, 6thJuly 2023

Claims

CLAIMS Method for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents characterized by, comprising a step of: assessing the level of a-tubulin acetylation in cancer cells. Composition for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents by assessing the presence or amount of a-tubulin acetylation in cancer cells in which the said composition is characterized by, comprising a reagent to detect acetylated a-tubulin, a non-limiting example of which comprises antiacetylated a-tubulin (acetyl K40) antibody ab24610 from clone 6-11B-1 . Kit for predicting prognosis of treatment with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents characterized by, comprising the composition described in claim 2 and the means for determining the presence or amount of a-tubulin acetylation in cancer cells, including controls. Method for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents for a patient characterized by, comprising the steps of : a) assessing the presence or amount of a-tubulin acetylation in cancer cells and; b) adjusting the treatment regimen in regard to dose, infusion time, number of treatment cycles, duration of treatment cycles, duration of intervals between treatment cycles, overall duration of treatment, and combinations thereof. Composition for deriving an adjusted treatment regimen with taxol, taxanes or other microtubule (MT) stabilizing anti-cancer agents for a patient through assessing the presence or amount of a-tubulin acetylation in cancer cells in which the said composition is characteri zed by, comprising a reagent to detect acetylated a-tubulin, a non-limiting example of which comprises anti-acetylated a-tubulin ( acetyl K40 ) antibody ab24610 from clone 6- 11B- 1 . Kit for deriving an adj usted treatment regimen with taxol , taxanes or other microtubule (MT ) stabili zing anti-cancer agents for a patient characteri zed by, comprising the composition described in claim 5 and the means for determining the presence or amount of a-tubulin acetylation in cancer cells , including controls .Lisbon, 6thJuly 2023

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