Figure 4 from Targeting Transcriptional Regulation with a CDK9 Inhibitor Suppresses Growth of Endocrine- and Palbociclib-Resistant ER<sup>+</sup> Breast Cancers
Authors: Soosainathan Arany , Iravani Marjan , El-Botty Rania , Alexander John , Sourd Laura , Morisset Ludivine , Painsec Pierre , Orha Rebecca , Nikitorowicz-Buniak Joanna , Pancholi Sunil , Haider Syed , Dowsett Mitch , Marangoni Elisabetta , Martin Lesley-Ann , Isacke Clare M. 
Published: January 2024

<p>CDK9 inhibitor AZD4573 drives tumor regression in endocrine therapy and palbociclib-resistant PDXs. <b>A,</b> 2–5 × 10<sup>4</sup> dissociated PDO cells were seeded per well in 96-well tissue culture plates in 10% Matrigel. The resulting organoids were treated with escalating doses of AZD4573 at day 2. Cell viability was assessed on day 7. Dose–response curves showing percentage of viable AZD4573-treated cells compared with vehicle control. <i>n</i> = 4 wells per PDO. Drug response represented by sigmoidal dose–response curve. IC<sub>50</sub> and AUC (area under curve) values are shown. ND, not determined. <b>B</b> and <b>C,</b> HBCx-180 (<b>B</b>) and HBCx-134-palboR31 (<b>C</b>) PDXs were inoculated into 8-week-old Swiss nude mice. Xenografts were randomly assigned to different groups when tumors reached a volume of 100 to 200 mm<sup>3</sup> and treated with vehicle, palbociclib + fulvestrant, AZD4573 or the combination of palbociclib + fulvestrant + AZD4573 (V, P+F, A, P+F+A). Top, relative tumor volumes ±SD. HBCx-180, <i>n</i> = 6–7; HBCx-134-palboR31, <i>n</i> = 7–10 mice per group (two-sided Mann–Whitney <i>U</i> test). Bottom, waterfall plots showing percent change in tumor volume from baseline. HBCx-180, <i>n</i> = 5–7; HBCx-134-palboR31, <i>n</i> = 7–8 mice per group. <b>D–G,</b> RNA-seq analysis of HBCx-134-palboR31–treated tumors<b>. D,</b> Number of genes differentially up- and downregulated in tumor treatment group comparisons, using significance thresholds of |log<sub>2</sub>FC| > 1 and FDR adjusted <i>P</i> < 0.05. <b>E,</b> Expression of <i>MYC</i>, <i>MYB</i>, and <i>MCL1</i> in tumor treatment groups. <i>P</i> values were estimated using edgeR's implementation of quasi-likelihood F test. <b>F,</b> Heat map of genes with synergistic downregulated expression in palbociclib + fulvestrant + AZD4573–treated tumors (log<sub>2</sub>FC > 1, FDR-adjusted <i>P</i> < 0.05). <b>G,</b> Expression of <i>MYCN</i> and <i>WEE1</i> in tumor treatment groups. <i>P</i> values were estimated using edgeR's implementation of quasi-likelihood F-test. <b>H,</b> 5,000 MCF7 LTED<sup>Y537C</sup> or T47D LTED (L) or LTED<sup>PalboR</sup> (LP) cells were seeded in 96-well ultra-low attachment round-bottomed plates and resulting spheroids treated with AZD4573 (10 nmol/L) or palbociclib (1 μmol/L) or combination at days 3 and 6. Cell viability was assessed on day 10. Data represents relative cell viability compared with vehicle control (<i>n</i> = 4 biological replicate, <i>n</i> = 5 technical replicates per biological replicate; mean values ±SEM; two-way ANOVA with Sidak multiple comparisons test, and confidence intervals of 95%). AZD, AZD4573; Combi, AZD4573 + palbociclib; Palbo, palbociclib; Veh, vehicle.</p>