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'CDK4/6 inhibitors' targeting retinoblastoma (Rb) protein to block cell cycling emerges as the leading drug for treating advanced or metastatic breast cancer and the significance of biomarkers

October is the breast cancer (affects ~13% of women in the U. S.) awareness month and we have come a long way as 5-year survival rate for localized and regional cases (cancer cells spread to nearby lymph nodes) are approaching ~99% and >86%, respectively (albeit lesser for 10-year survival rate).  Nevertheless, out of >270,000 women (and >2,600 men) who are expected to develop breast cancer in 2020 in the U. S. alone, ~42,000 are projected to die--mainly due to metastatic breast cancer (5-year survival rate of 26%) which remains largely incurable. (https://www.healthline.com/health/breast-cancer/survival-facts-statistics#by-stage). Approximately 3.8 million women in the U. S. are diagnosed with breast cancer presently.  For breast cancer, common metastatic sites include brain, lung, liver, and bone. 

The current management of breast cancer is guided by their classification based on the expression pattern of specific receptors.  For Her2-positive cancers, treatments include Herceptin (Trastuzumab), an antibody targeting Her2 receptor overexpressed in ~20-30% of all breast cancers.  For hormone-positive cancers (70-80%) expressing estrogen receptor (ER) and/or progesterone receptor (PR), treatments include 'hormone therapy' (more accurately, anti-hormone therapy) targeting estrogen receptor (ex. antagonist, tamoxifen) or its biosynthesis pathway (ex. aromatase inhibitor, letrozole).  For 'triple negative' breast cancers (express little or none of the above 3 receptors; 10-20%), treatment options are limited except for the standard treatments such as chemotherapy, surgery and radiotherapy.

At the latest American Association of Cancer Research (AACR)-sponsored international breast cancer symposium held in December 2019 in San Antonio, the major part of the sessions was focused on the latest clinical results obtained using the “CDK inhibitor”.  These targeted drugs include the (chemical) drugs Palbociclib (Pfizer), Ribociclib (Novartis), and Abemaciclib (Eli Lilly) and multiple other CDK inhibitors currently undergoing clinical trials. 

Previous studies showed that, when combined with anti-hormone therapy, Palbocicclib could nearly double the 'progression-free survival' (refers to the period before conditions worsen), i.e.~14.5 months with anti-hormone therapy alone versus ~24.8 months with the combination therapy (Finn et al., 2016).  Further, the recent studies showed that the treatment was also effective in breast cancer patients who were heavily pre-treated (Serra et al., 2019).  Encouraged by the data, the U. S. Food and Drug Administration rapidly approved CDK inhibitors for the treatment of hormone receptor-positive (but Her2 negative) advanced-stage or metastatic breast cancer.  Clinical trials have now been expanded to examine the efficacy of CDK inhibitors in other subsets of breast cancer (ex. Her2 positive) as well as other types of human cancers such as melanoma and leukemia.



      

Briefly, the development of CDK inhibitors was preceded by molecular genetics research investigating the genetic basis of human cancers.  Central to the undertaking was the delineation of 'two-hit hypothesis' proposed by late A. Knudson (Fox Chase Cancer Center, USA), who predicted that the loss of both alleles of a ‘tumor suppressor gene’ may predispose to retinoblastoma. Retinoblastoma is a childhood cancer of the retina (eye), which may occur in hereditary or sporadic manner.  The hypothesis was confirmed by the identification of human RB gene by several research groups (Lee et al., 1987; Friend et al., 1986).  Using the cloned gene, the mutational loss of RB gene in retinoblastoma was documented.

Subsequently, researchers showed that the cloned RB gene could suppress the growth of human cancer cell lines in vitro or tumors grown in animal models in vivo.  This has inspired a great interest by pharmaceutical industries in exploiting the growth inhibitory property of RB to suppress cancer.

The normal cell cycle consists of G1, S (DNA replication), G2 and M (chromosome segregation into daughter cells) phases.  "Restriction point" refers to a point in G1 (several hours before S phase), after which cells are "committed" to complete the rest of cell cycle.  Cancer cells acquire the ability to divide uncontrollably by deregulating the mechanism controlling the restriction point.  Furthermore, all signaling pathways activated by growth factors or hormones converge at the restriction point to modulate cell growth.

Interestingly, microinjection of purified Rb protein led to G1 arrest (Goodrich et al., 1991).  'DNA damage checkpoint' refers to a built-in cellular mechanism that blocks cell cycling to allow time for repair in the event of DNA damage.  Subsequently, investigators at the Johns Hopkins University (USA) showed that RB is a component of G1 checkpoint that arrests cell cycle at G1 (to repair damaged DNA before proceeding with DNA replication in S phase) (Slebos et al. ,1994).  These findings revealed that RB regulates the restriction point to control passage across G1, thus assuming a central role in cell cycle control. 

Rb is a nuclear protein that becomes increasingly phosphorylated as cells progress from G1 to M phase (Lee et al., 1987).  It indicated that the least phosphorylated (hypo-phosphorylated) RB species found in G1 represents the functionally active form.  Rb is considered a 'master regulator' affecting the transcription of >200 genes to control G1-to-S progression (Dyson et al., 2018).

Cell cycle research using yeasts have shown that progression through each cell phase is controlled by distinct cyclin-dependent kinases (CDKs).  CDKs are serine/threonine kinases, whose activity requires forming a complex with various cyclins.  Intriguingly, Rb was found to be phosphorylated by various cyclin-CDK complexes (see Figure).  In early G1 phase, Rb is phosphorylated by CDK4 or CDK6 complex.  Consequently, pharmaceutical industries have endeavored to find inhibitors of these kinases (to keep RB hypo-phosphorylated), which resulted in the discovery of CDK 4/6 inhibitors mentioned above. In the era of COVID-19, there has been an increase in the cancer-associated mortality due to avoidance of hospital visits by patients, making it more urgent to develop pertinent biomarkers to monitor therapy progress as well as to identify the responsive patients on an outpatient basis.

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References

Dyson NJ et al. Non-canonical functions of the RB protein in cancer.  Nat Rev Cancer. 18:442-451 (2018). PMID: 29692417

Finn RS, Martin M, Rugo HS, et al.  Palbociclib and Letrozole in Advanced Breast Cancer.  N Engl J Med. 375:1925-1936 (2016).  PMID: 27959613

Friend SH, Bernards R, Rogelj S, Weinberg RA, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma.  Nature. 323:643-6 (1986). PMID: 2877398

Goodrich DW, Lee WH, et al. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.  Cell  67:293-302 (1991).  PMID: 1655277

Lee WH, Bookstein R, Hong F, et al. Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science. 235:1394-9 (1987).  PMID: 3823889

Lee WH, Shew JY, Hong FD, et al. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity.  Nature. 329:642-5 (1987).  PMID: 3657987

Serra F, Lapidari P, Quaquarini E, et al. Palbociclib in metastatic breast cancer: current evidence and real-life data.  Drugs Context. 8:212579 (2019).  PMID: 31391852

Slebos RJ, , Jacks T, Kastan MB, et al. p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein.  Proc Natl Acad Sci USA 91:5320-4 (1994). PMID: 8202487