Additional file 4 of Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax
Creators: name: Gaur, Tarang
name: Poddutoori, Ramulu
name: Khare, Leena
name: Bagal, Bhausaheb
name: Rashmi, Sonal
name: Patkar, Nikhil
name: Tembhare, Prashant
name: PG, Subramanian
name: Shetty, Dhanlaxmi
name: Dutt, Amit
name: Zhang, Qi
name: Konopleva, Marina
name: Platzbeckar, Uwe
name: Gupta, Sudeep
name: Samajdar, Susanta
name: Ramchandra, Murali
name: Khattry, Navin
name: Hasan, Syed K.
Publisher: name: figshare
Year: 2023
Subjects: Biophysics
Biochemistry
Cell Biology
Molecular Biology
Pharmacology
Cancer
Hematology

description: Additional file 4: Supplementary Figure 1. Change in protein expression of CDK7 and RPII after XL102 treatment- Quantification of Western blot data at 6hrs and 24hrs of drug treatment. Results are the mean ± SD of three independent experiments. P values &lt; 0.05 were considered significant, where p value &lt; 0.05 (∗), p value &lt; 0.01(∗∗), p value &lt; 0.001 (∗∗∗), p value &lt; 0.0001 (∗∗∗∗). Supplementary Figure 2. XL102 treatment leads to apoptosis: (A) The levels of pro-apoptotic and anti-apoptotic protein was quantified after 24hrs of XL102 treatment in MOLM13 as well as OCI AML2 cell lines. (B) The levels of MCL1, XIAP and BCL-XL in patient derived AML blast were quantified after 24hrs of XL102 treatment. Supplementary Figure 3. CDK7 depletion leads to modulation of cell cycle: (A) The levels of phospho CDK1and CDK2 decreases in dose dependent manner after 6hrs and 24hrs of treatment. (B) CFSE staining of leukemic cells after 24hrs of XL102 treatment. Geometric mean or mean fluorescence intensity of dye within the cells increases on drug treatment indicating proliferation arrest. (C) Protein quantification of c-Myc, p21,p27, CyclinD and CDK7 in c-MycKO cells versus control. (D) Protein quantification of CDK7, p53, p21, p27 and Ser2/5/RPII in CDK7KO versus control (E) Though the proliferation rate in CDK7KO cells in comparison to MOLM13 cells was slightly decreased, there was no statistical difference (p=0.48, n=3). Supplementary Figure 4. Effect of XL102 treatment on AML animal models (A) Representative image of H&amp;E-stained tumor sample from each group after 12 days of drug treatment (B) Change in body weight of animals in different groups. (C) Representative image of confirmation of engraftment using dot plot of mCD45 and hCD45 in peripheral blood and bone marrow of animals. (D) Percentage changes in body weight of animals in different groups. Supplementary figure 5. Combination of XL102 with Venetoclax shows synergy in AML cells. (A) Pearson’s correlation matrix based on the gene expression showing the exclusiveness of all four conditions (Parental untreated, parental treated, resistant untreated and resistant treated). There is a higher correlation found between the parental and resistant samples respectively. (B) MA plot representing differential analysis of the three comparisons (Parental untreated vs resistant untreated, parental untreated vs parental treated and resistant untreated vs resistant treated) and highlighting the significantly up or down-regulated genes in each of them. (C) GSEA analysis of VenS cells showing differentially regulated genes. (D) Q-PCR analysis of modulated genes after XL102 treatment for 24hrs. (E) The combinatorial treatment of XL102 with Venetoclax shows synergy in OCI AML2 cells as the combination index values were less than 1 in all combinations. (F) Enhanced apoptosis was observed after a combination of XL102 and Venetoclax using Annexin/Pi staining in AML cells. (G) Western blot quantification of basal level expression of apoptotic proteins and p53 in MOLM13 Venetoclax sensitive versus resistant cells (H) Quantification of western blots of apoptosis protein after combination of XL102 and Venetoclax in MOLM13 Venetoclax resistant cells. (I-J) Determination of apoptosis markers from tumor excised from xenografts and the quantification of blots.