Potent Anti-Leukemic Activity of a Novel Cdk9 Inhibitor in Preclinical CLL Mouse Models
Authors: Göthert Joachim Rudolf , Imsak Roze , Möllmann Michael , Weber Stefanie , Necke Mandy , Dührsen Ulrich , Eickhoff Jan , Baumann Matthias , Unger Anke , Schultz-Fademrecht Carsten , Dürig Jan 
Published: October 2019

Abstract 1789

Active agents for the treatment of CLL are available. However, CLL eventually progresses to refractory disease. Defects in apoptosis, cytogenetic abnormalities and alterations in non-malignant cells of the micro-environment are likely to be the major causes of therapeutic resistance. Therefore, novel agents are required to abrogate apoptosis blocks and to alter the micro-environment of CLL. One approach targets cyclin-dependent kinases (Cdk). Specifically, Cdk9 promotes initiation and elongation steps in transcription maintaining expression levels of anti-apoptotic proteins with inherently rapid turnover rates. Inhibitors of cyclin-dependent kinases (Cdks) have been previously shown to exert cytotoxic effects on CLL cells by inhibiting Cdk7 and Cdk9. However, inhibitors used in these studies suffer from lack of selectivity, making unambiguous interpretation of results difficult, and leading to unspecific toxicity.

Here, we studied the novel Cdk inhibitor LDC-9-A, which exhibits potent and selective inhibitory activity against Cdk9. The in vitro cytotoxicity of LDC-9-A was evaluated after 24 and 48 hours in CLL cases (n=8) and the CLL derived prolymphocytic cell line MEC-1. After a culture period of 48 hours LD50 values ranged between 0.1 and 1.9 μM for primary CLL cells and MEC-1 cells, respectively. Western blot analysis of protein extracts from MEC-1 cells showed that LDC-9-A down-modulates the anti-apoptotic proteins Bcl-2 and Mcl-1 in a dose- and time-dependent manner.

In order to study the in vivo anti-CLL activity of LDC-9-A 9–12 month-old transgenic TCL1 mice (n=4) were treated for two consecutive days with an orally bioavailable LDC-9-A formulation (50 mg/kg body weight). Control groups consisted of TCL1 mice receiving vehicle only (n=3) and wild-type control littermates (n=4) receiving LDC-9-A. Numbers of peripheral blood leukemic cells (CD19+CD5+CD3−) and non-malignant T-cells (CD19−CD5+CD3+) were longitudinally evaluated by flow cytometry. Remarkably, LDC-9-A treatment of TCL1 transgenic mice led to a decrease of blood leukemic cells to 6.3±2.0% (mean±SEM) of baseline levels (p<0.0001) two days post treatment cessation. The numbers of T-cells decreased to 15.5±6.6% (p<0.01) in TCL1 mice while the blood T-cells of LDC-9-A treated wild-type controls did only decrease to 49.5±11.6% of baseline levels. Because LDC-9-A was expected to alter cellular Cdk9-mediated anti-apoptotic protein replenishment Bcl-2 levels were determined by flow cytometry analysis. Interestingly, cellular Bcl-2 levels of surviving leukemic cells as well as surviving non-malignant T-cells were significantly higher compared to levels before treatment (1.6- and 2.6-fold, respectively, p<0.05). These data implied that either LDC-9-A was not able to clear cells with high Bcl-2 expression or that LDC-9-A led to a compensatory up-regulation of Bcl-2 in residual cells.

Finally, the anti-CLL activity of LDC-9-A was further validated by employing a recently developed adoptive transfer model of primary human CLL cells. In this model NOD/SCID/γcnull (NSG) mice were transplanted with CLL cells and treated with different doses of LDC-9-A for 3–5 sequential days. In line with the data generated with the TCL1 murine CLL model the LDC-9-A treatment of xenografted animals with 60 and 90 mg/kg body weight resulted in an 80–90% reduction of human CLL cell recovery from murine spleens as compared to vehicle treated controls.

In summary, we demonstrated effective anti-CLL activity of the novel Cdk9 inhibitor LDC-9-A in two independent preclinical mouse models. The activity of LDC-9-A against CLL cells was achieved at least in part by modulating cellular anti-apoptotic protein levels. Furthermore, LDC-9-A interfered with non-malignant T-cells, which represent an important component of the CLL micro-environment.

Disclosures:

Eickhoff: Lead Discovery Center GmbH (LDC): Employment. Baumann:Lead Discovery Center GmbH (LDC): Employment. Unger:Lead Discovery Center GmbH (LDC): Employment. Schultz-Fademrecht:Lead Discovery Center GmbH (LDC): Employment. Dürig:Roche: Speakers Bureau; GSK: Speakers Bureau; Santaris Pharma: Consultancy, Research Funding; Celgene: Research Funding.