The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

Abstract

CDK4/6 inhibitors arrest the cell cycle in G1 and are used in combination with hormone therapy to treat advanced HR+/HER- breast cancer. To allow more effective use of these drugs in breast cancer, and to facilitate their use in other tumour types, biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify the most sensitive tumour types and genotypes have misrepresented the responsive cell lines because of a reliance on ATP-based proliferation assays. When cells arrest in G1 following CDK4/6 inhibition, they continue to grow in size, producing more mitochondria and ATP. This cellular overgrowth masks an efficient arrest using metabolic ATP-based assays, but not if DNA-based assays are used instead. By comparing tumour cells using different assay types, we demonstrate that the lymphoma lines previously identified as the most responsive cell types, simply appear to respond the best because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib, but this is because it also inhibits cell overgrowth through off-target effects. DepMap analysis of previous screening data using only the reliable assay types, demonstrates that palbociclib-sensitivity is associated with sensitivity to Cyclin D1, CDK4 and CDK6 knockout/knockdown, and resistance is associated with sensitivity to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Furthermore, potential biomarkers of palbociclib-sensitivity are increased expression of Cyclin D1 (CCND1) and RB1, and reduced expression of Cyclin E1 (CCNE1) and CDKN2A. None of these associations are present when analysing DepMap using similar data from metabolic assays. This reinforces the importance of new screens to assess CDK4/6 inhibitors against a wide range of cancer cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.

Too big not to fail: emerging evidence for size‐induced senescence

“…Based on this observation, excess cell size is predicted to severely compromise mitochondrial function. Consistent with this prediction, prolonged Cdk4/6 inhibition increases mitochondrial mass [81,85,86] but also causes an accumulation of mitochondria‐derived ROS [85,87,88], suggesting mitochondrial dysfunction. Moreover, excessively large cells upregulate SOD2—which neutralizes superoxide species—suggesting an increased need to neutralize ROS [21,71].…”

“…Many cell cycle arrest-causing drugs that are either currently used for cancer therapies or are in clinical trials (e.g., doxorubicin, Cdk4/6 inhibitors, Cdk7 inhibitors, etoposide) have been reported to drastically increase cell size in vitro [19,20,[22][23][24]81,124]. Still, this is not true in all cultured cell lines: many blood cancers are reported to maintain their size in response to the Cdk4/6 inhibitor palbociclib [21,81]. A list of human cell lines where size has been evaluated upon multi-day Cdk4/6 inhibition is included in Table 1.…”

“…Recent studies demonstrate that the suppression of biomass accumulation (e.g., by mTOR inhibition, contact inhibition, or serum starvation [19,21,22,26,81,126]) in combination with drugs that cause cell cycle arrest rescues long-term proliferation in vitro. This has been demonstrated in the context of palbociclib [19,21,22,24,25], the MDM2 inhibitor nutlin3a [21,127], etoposide [52], and doxorubicin [52].…”

“…Mitochondrial content increases with respect to cell size [80–82]. Indeed, mitochondrial DNA content has been shown to scale with cell volume rather than nuclear genome content in yeast [83,84], demonstrating an intrinsic relationship between cell size and mitochondrial abundance.…”

Improving the power of drug toxicity measurements by quantitative nuclei imaging

“…The difference between measurements performed by different methods also depended on a drug selection, especially for drugs that cause cell cycle arrest or other cytostatic drugs as has been shown before 18,36 . For example, palbociclib is known to induce cell size growth 37 and accumulation of mitochondria, thus resulting in false results obtained by methods relying on mitochondrial activity 38 . We showed that palbociclib as well as other cell cycle inhibitor not only induce mitochondria accumulation due to increased cell size, but also increase mitochondrial activity itself.…”