COMPOSITIONS AND METHODS INVOLVING ENGINEERED P27

This application claims priority to U.S. Provisional Application No. 62/663,914, filed Apr. 27, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

This invention was made with government support under Grant Nos. CA132685 and CA206244, awarded by the National Institutes of Health. The government has certain rights in this invention.

Cyclin-dependent kinases (Cdk) 4 and 6 promote cell proliferation through their kinase activity. Inhibitors of Cdk4 and Cdk6 may function as cancer therapeutics. The active cellular form of the enzyme Cdk4 or Cdk6 is in complex with cyclin D (CycD) and p27. Current inhibitors of Cdk4/6 were developed using Cdk4-CycD dimeric complexes that lack p27, in part because of the technical challenges in generating the active form of p27. However, the Cdk4-CycD dimeric complex does not readily form in all cells. Further, certain complexes including p27 may be resistant to treatments.

In one aspect, the disclosure features a polypeptide comprising an engineered p27, or a fragment thereof, wherein the engineered p27 has at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89, wherein the engineered p27 forms a trimeric protein complex with (i) a cyclin-dependent kinase 4 (Cdk4) or a variant thereof, or a Cdk6 or a variant thereof, and (ii) a cyclin D (CycD) or a variant thereof, and wherein the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1.

In some embodiments, the engineered p27 comprises amino acid substitution Y74E or Y74D. In some embodiments, the engineered p27 comprises amino acid substitution Y74E, Y74D, or Y74R. In some embodiments, the engineered p27 comprises amino acid substitution Y88E or Y88D. In some embodiments, the engineered p27 comprises amino acid substitution Y89E or Y89D.

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1.

In some embodiments of this aspect, the engineered p27 comprises a sequence of KPSACRNLFGPVDHEELTRDLEKHCRDMEEASQRKWNFDFQNHKPLEGKX₁EWQEVE KGSLPEFX₂X₃RPPRPPKGA (SEQ ID NO: 59), wherein X₁is Y, E, D, or R; X₂is Y, E, or D; and X₃is Y, E, or D, and wherein at least one of X₁, X₂, and X₃is not Y. In some embodiments, X₁is Y. In some embodiments, X₁is E. In some embodiments, X₁is D. In some embodiments, X₁is R. In some embodiments, X₂is Y. In some embodiments, X₂is E. In some embodiments, X₂is D. In some embodiments, X₃is Y. In some embodiments, X₃is E. In some embodiments, X₃is D.

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the engineered p27 comprises a sequence having at least 90% sequence identity to the sequence of

In another aspect, the disclosure features a trimeric protein complex comprising: (i) a polypeptide comprising an engineered p27 as described in the previous aspect, or a phosphorylated, wild-type p27 or a fragment thereof (ii) a Cdk4 or a variant thereof, or a Cdk6 or a variant thereof and (iii) a CycD or a variant thereof, wherein the Cdk4 or the variant thereof or the Cdk6 or the variant thereof in the trimeric protein complex is an active kinase.

In some embodiments of this aspect, the Cdk4 or the variant thereof or the Cdk6 or the variant thereof is capable of phosphorylating a protein comprising a phosphorylation site having the sequence X₁PX₂X₃(SEQ ID NO: 60), wherein X₁is S or T; X₂is any amino acid; and X₃is K or R. In some embodiments, the protein comprising the phosphorylation site is retinoblastoma protein (Rb), FoxM1, or histone H1.

In some embodiments of this aspect, the CycD is CycD1, CycD2, CycD3, or a variant thereof.

In some embodiments of this aspect, the CycD1 or the variant thereof comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the CycD2 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the CycD3 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the Cdk4 or a variant thereof comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the variant of Cdk4 comprises T172E or T172D, and the amino acid positions are determined with reference to the sequence of SEQ ID NO: 37. In some embodiments, the variant of Cdk4 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the variant of Cdk4 has amino acid residues 44 to 46 deleted, G43E or G43D, and G47E or G47D, and the amino acid positions are determined with reference to the sequence of SEQ ID NO: 37. In some embodiments, the variant of Cdk4 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the variant of Cdk4 has amino acid residues 44 to 46 deleted, G43E or G43D, G47E or G47D, and T172E or T172D, wherein the amino acid positions are determined with reference to the sequence of SEQ ID NO: 37. In some embodiments, the variant of Cdk4 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the Cdk6 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the variant of Cdk6 comprises T177E or T177D, wherein the amino acid positions are determined with reference to the sequence of SEQ ID NO: 52. In some embodiments, the variant of Cdk6 comprises a sequence having at least 90% sequence identity to the sequence of

In some embodiments of this aspect, the phosphorylated, wild-type p27 or a fragment thereof comprises the sequence of any one of SEQ ID NOS: 1-3 and is phosphorylated at Y74, Y88, and/or Y89, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1.

In another aspect, the disclosure features a method of screening for inhibitors of a trimeric protein complex comprising an active Cdk4 or a variant thereof, or an active Cdk6 or a variant thereof, comprising: (a) providing a trimeric protein complex described in the previous aspect; (b) contacting the trimeric protein complex with a compound and a substrate of the Cdk4 or the variant thereof or the Cdk6 or the variant thereof and (c) determining the phosphorylation status of the substrate, wherein the compound is an inhibitor of the trimeric protein complex if the compound inhibits the phosphorylation activity of the Cdk4 or the variant thereof or the Cdk6 or the variant thereof.

In some embodiments of this aspect, the method further comprises, prior to step (a), phosphorylating a wild-type p27 or a fragment thereof by contacting the wild-type p27 with a kinase. In some embodiments, the kinase is selected from the group consisting of Brk kinase, Src kinase, and Abl kinase.

In some embodiments of this aspect, the method further comprises, after step (c), comparing the phosphorylation status of the substrate with the phosphorylation status of the substrate when the compound is not present.

In some embodiments of this aspect, the substrate comprises a phosphorylation site having the sequence X₁PX₂X₃(SEQ ID NO: 60), wherein X₁is S or T; X₂is any amino acid; and X₃is K or R. In some embodiments, the substrate comprising the phosphorylation site is Rb, FoxM1, histone H1, or a variant thereof.

In another aspect, the disclosure features a method of expressing and purifying a trimeric protein complex comprising (i) a phosphorylated, wild-type p27 or a fragment thereof (ii) a Cdk4 or a variant thereof or a Cdk6 or a variant thereof and (iii) a CycD or a variant thereof, the method comprising: (a) expressing the Cdk4 or the variant thereof or the Cdk6 or the variant thereof, and the CycD or the variant thereof in a first cell line, where the first cell line comprises one or more expression vectors configured to express the Cdk4 or the variant thereof or the Cdk6 or the variant thereof, and the CycD or the variant thereof (b) expressing the wild-type p27 or the fragment thereof in a second cell line, where the second cell line comprises an expression vector configured to express the wild-type p27 or the fragment thereof (c) purifying the Cdk4 or the variant thereof or the Cdk6 or the variant thereof, and the CycD or the variant thereof from the first cell line and the wild-type p27 or the fragment thereof from the second cell line; (d) phosphorylating the wild-type p27 or the fragment thereof obtained from step (c) with a kinase; and (e) combining the phosphorylated, wild-type p27 or the fragment thereof obtained from step (d) with the purified Cdk4 or the variant thereof or the purified Cdk6 or the variant thereof, and the CycD or the variant thereof obtained from step (c) under conditions that allow the formation of the trimeric protein complex, wherein the Cdk4 or the variant thereof or the Cdk6 or the variant thereof in the trimeric protein complex is an active kinase.

In some embodiments of this aspect, the expression vector is a baculovirus vector. In some embodiments, the first and/or second cell line is an insect cell line.

Genetic and biochemical studies have demonstrated that the retinoblastoma protein (Rb) pathway is a major regulator of cell cycle progression in G1 phase^{1, 2}. In G0/G1 phase, Rb and its family members p107 and p130 inhibit the E2F family of transcription factors (e.g., E2F1-5). In response to mitogenic signals, cyclin-dependent kinase (Cdk)-cyclin complexes phosphorylate Rb family members, which results in the disruption of complexes between Rb and E2F family members and allows the transcription of genes essential for S-phase progression. Cdk-cyclin complexes, e.g., Cdk4/6-CycD and Cdk2-CycE/A, are inhibited by proteins from the p16 family and can be either inhibited or activated by proteins from the p27 (p21, p27, p57) family.

With the goal of preventing Rb inactivation and cancer cell-cycle progression, specific inhibitors of Cdk4 and/or Cdk6 have been developed in the past decade. These inhibitors were found in screens against recombinant Cdk4-CycD dimeric complex. One of these inhibitors, palbociclib, was approved in 2015 for the treatment of estrogen receptor-positive breast cancer^3-5. Several other Cdk4/6 inhibitors are being tested (e.g., ribociclib, abemaciclib, trilaciclib) in multiple cancer types^6-8. Key unresolved challenges limiting Cdk4/6 inhibitors are, e.g., mechanisms of inherent resistance, acquired resistance, and early adaptation.

The activity of p27 (also known as cyclin-dependent kinase inhibitor 1B) towards Cdk4/6 is complex. p27 inhibits Cdk4/6-CycD activity in vitro and in cells under conditions of growth arrest^9-13. At the same time, however, p27 increases Cdk4/6-CycD stability and is always present in active Cdk4/6-CycD complexes that phosphorylate Rb in proliferating cells^14-18. Phosphorylation of p27 by tyrosine kinases (e.g., Src kinase, Brk kinase, Abl kinase) on amino acid residues Y74, Y88, and Y89 of p27 further increases Cdk4/6 activity, and this phosphorylation has been suggested to switch p27 from an inhibitor to an activator^19-21.

Disclosed herein are the structure and activity of the p27-Cdk4/6-CycD complex. Also disclosed is a method of expressing and purifying an active, recombinant p27-Cdk4/6-CycD complex.

In some embodiments, the method involves treating p27 with an active kinase (e.g., tyrosine kinase) such as recombinant Brk, Src, or Abl kinases. In some aspects of this embodiment, the p27 is treated prior to assembly of the enzyme. In other embodiments, the method involves using a p27 polypeptide that comprises a mutation at Y74, a mutation at Y88, and/or a mutation at Y89, or any combination thereof. In some aspects of this embodiment, the p27 polypeptide comprises a Y74E mutation and no mutation at Y88 or Y89. In other aspects, the p27 polypeptide comprises a Y88E mutation and a Y89E mutation In other aspects, the p27 polypeptide comprises a Y74R mutation, a Y88E mutation, and a Y89E mutation. It is disclosed herein that p27-activated Cdk4-CycD complex: (1) has broader substrate specificity than the Cdk4-CycD dimeric complex and (2) is resistant to treatment of palbociclib. For these reasons, the p27-Cdk4/6-CycD enzyme complex may be used for screening of new inhibitors that are effective in different cancer types.

As used herein, the term “engineered p27” refers to a p27 polypeptide that contains one or more amino acid substitutions, additions, and/or deletions relative to the amino acid sequence of a wild-type p27 (e.g., SEQ ID NO: 1). An engineered p27 may have the same length as a wild-type p27 or may be a fragment of the wild-type p27. An engineered p27 as described herein may have at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1. Further, an engineered p27 as described herein forms a trimeric protein complex with a cyclin-dependent kinase 4 (Cdk4) or a variant thereof, or Cdk6 or a variant thereof, and a cyclin D (CycD) or a variant thereof.

As used herein, the term “Cdk4 or a variant thereof” refers to a wild-type cyclin-dependent kinase 4 (Cdk4) or a variant of the wild-type Cdk4. A wild-type Cdk4 may have the sequence of SEQ ID NO: 37. A variant of the wild-type Cdk4 (also called Cdk4 variant) refers to a Cdk4 that contains one or more amino acid substitutions, additions, and/or deletions relative to the amino acid sequence of the wild-type Cdk4 (e.g., SEQ ID NO: 37). A Cdk4 variant may have the same length as a wild-type Cdk4 or may be a fragment of the wild-type Cdk4. A Cdk4 variant as described herein is capable of phosphorylation activity and can form a trimeric complex with a CycD or a variant thereof, and an engineered p27 or a wild-type p27. An active Cdk4 or a variant thereof as used herein refers to a Cdk4 or a variant thereof that is an active kinase and is capable of phosphorylating at a phosphorylation site, e.g., a phosphorylation site having the sequence X₁PX₂X₃(SEQ ID NO: 60), wherein X₁is S or T; X₂is any amino acid; and X₃is K or R.

As used herein, the term “Cdk6 or a variant thereof” refers to a wild-type cyclin-dependent kinase 6 (Cdk6) or a variant of the wild-type Cdk6. A wild-type Cdk6 may have the sequence of SEQ ID NO: 52. A variant of the wild-type Cdk6 (also called Cdk6 variant) refers to a Cdk6 that contains one or more amino acid substitutions, additions, and/or deletions relative to the amino acid sequence of the wild-type Cdk6 (e.g., SEQ ID NO: 52). A Cdk6 variant may have the same length as a wild-type Cdk6 or may be a fragment of the wild-type Cdk6. A Cdk6 variant as described herein is capable of phosphorylation activity and can form a trimeric complex with a CycD or a variant thereof, and an engineered p27 or a wild-type p27. An active Cdk6 or a variant thereof as used herein refers to a Cdk6 or a variant thereof that is an active kinase and is capable of phosphorylating at a phosphorylation site, e.g., a phosphorylation site having the sequence X₁PX₂X₃(SEQ ID NO: 60), wherein X₁is S or T; X₂is any amino acid; and X₃is K or R.

As used herein, the term “cyclin D (CycD) or a variant thereof” refers to a wild-type CycD or a variant of the wild-type CycD (also called CycD variant) that is capable of forming a trimeric protein complex described herein comprising an active Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof). A wild-type CycD may be a wild-type CycD1, CycD2, or CycD3. A trimeric protein complex describe herein may comprise any one of the CycD1, CycD2, CycD3, or a variant thereof described herein.

As used herein, the term “trimeric protein complex” or “trimeric complex” refers to a complex formed by three proteins: (i) an engineered p27 or wild-type p27; (ii) a Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof); and (iii) a cyclin D (CycD) (or a variant thereof).

As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid or nucleic acid residues of a candidate sequence that are identical to the amino acid or nucleic acid residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment). In some embodiments, percent sequence identity can be any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence if the sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence as determined using the methods described herein; preferably BLAST using standard parameters, as described below.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A comparison window includes reference to a segment of any one of the number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The BLAST and BLAST 2.0 algorithms are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid or nucleic acid sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid or nucleic acid sequence identity to, with, or against a given reference sequence) is calculated as follows:

100×(fraction of A/B)

where A is the number of amino acid or nucleic acid residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid or nucleic acid residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid or nucleic acid sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid or nucleic acid sequence identity of the reference sequence to the candidate sequence.

In particular embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid or nucleic acid residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid or nucleic acid residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

In response to mitogenic signals, complexes involving p27, Cdk4 or Cdk6, and cyclin D (CycD) phosphorylate retinoblastoma protein (Rb), leading to the transcription of genes essential for S-phase cell cycle progression. In order to prevent Rb phosphorylation and cancer cell cycle progression, inhibitors of Cdk4 and Cdk6 have been developed in screens using a dimer of Cdk4 or Cdk6 and CycD due to technical challenges in generating the active form of p27 that can complex with Cdk4 or Cdk6 and CycD. However, the dimeric complex does not readily form in the cell. The protein p27 is always found together in complex with active Cdk4 or Cdk6 and CycD and may increase Cdk4/6-CycD stability. The disclosure features trimeric protein complexes comprising p27, Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof), and CycD, in which the Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof) in the trimeric protein complex is an active kinase. The trimeric protein complexes featured herein are closer mimics of the p27-Cdk4/6-CycD complexes found in vivo compared to the Cdk4/6-CycD dimeric complexes used in the past. The trimeric protein complexes described herein may serve as a better tool in screening and selecting chemical compounds that can function as inhibitors of the trimeric protein complex and Cdk4 or Cdk6 to prevent the phosphorylation of Rb, and accordingly, arresting cancer cell cycle progression.

In some embodiments, a trimeric protein complex described herein may comprise an engineered p27, a Cdk4 (or a variant thereof) or a Cdk6 (or a variant thereof), and a CycD (or a variant thereof), in which the Cdk4, Cdk6, or the variant thereof in the trimeric protein complex is an active kinase. The engineered p27 in the trimeric protein complex may have at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1. Examples of engineered p27 are provided in detail further herein.

In other embodiments, a trimeric protein complex may comprise a phosphorylated, wild-type p27, or a fragment thereof, a Cdk4 (or a variant thereof) or a Cdk6 (or a variant thereof), and a CycD (or a variant thereof), in which the Cdk4 (the variant thereof) or the Cdk6 (or the variant thereof) in the trimeric protein complex is an active kinase. In some embodiments, the phosphorylated, wild-type p27 or a fragment thereof comprises the sequence of any one of SEQ ID NOS: 1-3 and is phosphorylated at Y74, Y88, and/or Y89, wherein the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1. In order to form a trimeric protein complex with an active Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof), a wild-type p27 (or a fragment thereof) may be expressed from a separate cell line and phosphorylated by a kinase prior to formation of the trimeric protein complex.

The disclosure features an engineered p27 that can form a trimeric protein complex with a Cdk4 (or a variant thereof) or a Cdk6 (or a variant thereof), and a CycD (or a variant thereof). An engineered p27 as described herein may have at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1. In the trimeric protein complex, an engineered p27 may increase the stability of the dimer of Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof) and CycD (or a variant thereof). In some embodiments, an engineered p27 may have the same length as a wild-type p27 and contains at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89. In some embodiments, an engineered p27 may be a fragment of the wild-type p27 and contains at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89.

In some embodiments, an engineered p27 may have one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89. In some embodiments, an engineered p27 may have two amino acid substitutions at two positions selected from the group consisting of Y74, Y88, and Y89 (e.g., Y74 and Y88, Y74 and Y89, or Y88 and Y89). In some embodiments, an engineered p27 may have three amino acid substitutions at positions Y74, Y88, and Y89. In some embodiments, the amino acid substation at position Y74 may include, but are not limited to, Y74E and Y74D. In some embodiments, the amino acid substation at position Y74 may include, but are not limited to, Y74E, Y74D, and Y74R. The amino acid substitution at position Y88 may include, but are not limited to, Y88E and Y88D. The amino acid substitution at position Y89 may include, but are not limited to, Y89E and Y89D. In further embodiments, an engineered p27 may be phosphorylated, i.e., phosphorylated at a tyrosine residue (e.g., phosphorylated at one or more of Y74, Y88, and Y89).

Table 1 below lists the sequence of a wild-type p27, fragments of the wild-type p27, and various engineered p27 proteins containing at least one amino acid substitution at a position selected from the group consisting of Y74, Y88, and Y89, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 1. An engineered p27 described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the sequences of SEQ ID NOS: 1-36 listed in Table 1 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type p27 (SEQ ID NO: 1).

Cyclin-dependent kinase 4 or 6 (Cdk4 or Cdk6), when in complex with p27 and CycD, may act as an active kinase in phosphorylating Rb. The Cdk4 or Cdk6 in the trimeric protein complexes described herein may be a wild-type Cdk4 or a wild-type Cdk6, respectively. In other embodiments, the Cdk4 or Cdk6 in the trimeric protein complexes described herein may be a variant of the wild-type Cdk4 or the wild-type Cdk6, respectively, containing one or more amino acid substitutions, additions, and/or deletions relative to the wild-type protein sequence. A Cdk4 or Cdk6 variant may have the same length as the wild-type protein or may be a fragment of the wild-type protein. A Cdk4 variant or Cdk6 variant described herein is capable of phosphorylation activity and can form a trimeric complex with a CycD or a variant thereof, and an engineered p27.

Table 2 below lists the sequences of wild-type Cdk4 and Cdk6 and various Cdk4 and Cdk6 variants containing one or more amino acid substitutions relative to the wild-type protein, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 37 (Cdk4) or SEQ ID NO: 52 (Cdk6). A Cdk4 variant described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the sequences of SEQ ID NOS: 37-51 listed in Table 2 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type Cdk4 (SEQ ID NO: 37). A Cdk6 variant described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the sequences of SEQ ID NOS: 52-54 listed in Table 2 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type Cdk4 (SEQ ID NO: 52).

The CycD or a variant thereof in the trimeric protein complexes described herein may be a wild-type CycD or a variant of the wild-type CycD. A wild-type CycD may be a wild-type CycD1, CycD2, or CycD3. A CycD variant comprises one or more amino acid substitutions, additions, and/or deletions relative to the wild-type protein sequence (e.g., wild-type CycD1, CycD2, or CycD3). A trimeric protein complex comprising an active Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof) describe herein may comprise any one of CycD1, CycD2, CycD3, or a variant thereof described herein.

Table 3 below lists the sequences of wild-type CycD1, CycD2, CycD3, and various CycD variants containing one or more amino acid substitutions relative to the wild-type protein, in which the amino acid positions are determined with reference to the sequence of SEQ ID NO: 55 (CycD1), SEQ ID NO: 57 (CycD2), or SEQ ID NO: 58 (CycD3). A CycD1 variant described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 55 or 56 listed in Table 3 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type CycD1 (SEQ ID NO: 55). A CycD2 variant described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 57 listed in Table 3 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type CycD2 (SEQ ID NO: 57). A CycD3 variant described herein may have at least 90% sequence identity (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 58 listed in Table 3 and one or more amino acid substitutions, additions, and/or deletions relative to the wild-type CycD3 (SEQ ID NO: 58).

In some embodiments, for a trimeric protein complex comprising an engineered p27, a Cdk4 (or a variant thereof) or a Cdk6 (or a variant thereof), and a CycD (or a variant thereof), each member of the trimeric protein complex may be expressed from the same cell line or from separate cell lines. In some embodiments, all three members may be co-expressed from the same cell line, in which each member may be encoded in an expression vector configured to express the protein. In other embodiments, for a trimeric protein complex comprising a phosphorylated, wild-type p27 or a fragment thereof (e.g., any one of SEQ ID NOS: 1-3), a Cdk4 (or a variant thereof) or a Cdk6 (or a variant thereof), and a CycD (or a variant thereof), the wild-type p27, or a fragment thereof, may be expressed in a cell line separately from the other two members of the complex. Once the wild-type p27, or a fragment thereof, is isolated and purified, the wild-type p27, or a fragment thereof, may be incubated with a kinase (e.g., Brk kinase, Src kinase, and Abl kinase) in order to generate the phosphorylated wild-type p27 or fragment thereof. The phosphorylated, wild-type p27, or fragment thereof, may then be incubated with the Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof), and the CycD or a variant thereof, in order to generate the trimeric protein complex.

Each protein in the trimeric protein complex described herein may be produced from a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the proteins and complexes described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., insect or mammalian) origin.

Nucleic Acid Vectors and Host Cells

A nucleic acid sequence encoding the amino acid sequence of a protein (e.g., a engineered p27) may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. A nucleic acid molecule encoding a protein may be obtained using standard techniques, e.g., gene synthesis. Alternatively, a nucleic acid molecule encoding a wild-type protein (e.g., a wild-type p27 having the sequence of SEQ ID NO: 1) may be mutated to contain specific amino acid substitutions using standard techniques in the art, e.g., QuikChange™ mutagenesis. Nucleic acid molecules may be synthesized using a nucleotide synthesizer or PCR techniques.

Nucleic acid sequences encoding a protein in the trimeric protein complex of the disclosure (e.g., an engineered p27) may be inserted into a vector capable of replicating and expressing the nucleic acid molecules in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purpose of the disclosure. Each vector may contain various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding the protein of interest, and a transcription termination sequence. In some embodiments, a vector used to express a protein in the trimeric protein complex may be a baculovirus vector. In some embodiments, the baculovirus vector may have a polyhedrin promoter. In some embodiments, a vector used to express a protein in the trimeric protein complex may be a PGEX vector. In some embodiments, the PGEX vector may have a T7 promoter.

In some embodiments, insect cells are used as host cells for the disclosure. Examples of insect cells types include, but are not limited to, Sf9, Sf21, and S2 cells. In particular embodiments, Sf9 cells may be used to express a protein in the trimeric protein complex of the disclosure. In other embodiments, E. coli cells are used as host cells for the invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products. In other embodiments, mammalian cells are used as host cells for the invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the protein expressed. The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (Jul. 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (Jun. 28, 2012).

Protein Production, Recovery, and Purification

Host cells used to produce the proteins and complexes of the disclosure may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Host cells are cultured at suitable temperatures, such as from about 20° C. to about 39° C., e.g., from 25° C. to about 37° C., preferably 37° C., and CO₂levels, such as 5 to 10% (preferably 8%). The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression may be induced under conditions suitable for the activation of the promoter.

Protein recovery typically involves disrupting the host cell, generally by such means as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. The proteins may be further purified. A protein or complex of the disclosure may be purified by any method known in the art of protein purification, for example, by protein A affinity, other chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. (see Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009).

In some instances, a protein may be conjugated to a purification tag to facilitate purification and isolation of the protein from, e.g., a whole cell lysate mixture. In some embodiments, the purification tag binds to another moiety that has a specific affinity for the purification tag. In some embodiments, such moieties which specifically bind to the purification tag are attached to a solid support, such as a matrix, a resin, or agarose beads. Examples of purification tags that may be joined to a protein include, but are not limited to, a glutathione S-transferase (GST) tag and a hexa-histidine peptide (SEQ ID NO: 66). GST is a 211 amino acid protein (about 26 kDa) whose DNA sequence may be integrated into expression vectors for production of recombinant proteins. The result of expression from this vector is a GST-tagged fusion protein in which the functional GST protein may be fused to, e.g., the N-terminus or C-terminus of the recombinant protein. Because GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag may promote greater expression and solubility of recombinant proteins than expression without the tag. In addition, GST-tagged fusion proteins may be purified or detected based on the ability of GST to bind its substrate, glutathione (GSH). In some embodiments, a solid support may be functionalized with GSH to isolate and purified GST-tagged fusion proteins. A hexa-histidine peptide (HHHHHH (SEQ ID NO: 66)) binds to nickel-functionalized agarose affinity column with micromolar affinity. In some embodiments, the purification tag may be cleaved from the fusion protein once it is purified. A protease cleavage sequence (e.g., a TEV protease cleavage sequence ENLYFQG (SEQ ID NO: 67) may be inserted between the protein of interest and the purification tag.

In other embodiments, a FLAG peptide, a myc peptide, or a hemagglutinin (HA) peptide may be used as a purification tag. In some embodiments, a FLAG peptide includes the sequence DYKDDDDK (SEQ ID NO: 68). In some embodiments, a FLAG peptide includes integer multiples of the sequence DYKDDDDK (SEQ ID NO: 68) in tandem series, e.g., 3×DYKDDDDK (SEQ ID NO: 71). In some embodiments, a myc peptide includes the sequence EQKLISEEDL (SEQ ID NO: 69). In some embodiments, a myc peptide includes integer multiples of the sequence EQKLISEEDL (SEQ ID NO: 69) in tandem series, e.g., 3×EQKLISEEDL (SEQ ID NO: 72). In some embodiments, an HA peptide includes the sequence YPYDVPDYA (SEQ ID NO: 70). In some embodiments, an HA peptide includes integer multiples of the sequence YPYDVPDYA (SEQ ID NO: 70) in tandem series, e.g., 3×YPYDVPDYA (SEQ ID NO: 73). Antibodies that specifically recognize and bind to the FLAG, myc, or HA purification tag are well-known in the art and often commercially available. A solid support (e.g., a matrix, a resin, or agarose beads) functionalized with these antibodies may be used to purify a protein that includes a FLAG, myc, or HA peptide.

The disclosure also features methods of screening for inhibitors of the trimeric protein complexes described herein, which are closer mimics of the p27-Cdk4/6-CycD complexes found in vivo compared to the Cdk4/6-CycD dimeric complexes. The method comprises (a) providing the trimeric protein complex by incubating: (i) an engineered p27 described herein or a phosphorylated, wild-type p27 or a fragment thereof (e.g., any one of SEQ ID NOS: 1-36); (ii) a Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof); and (iii) a CycD or a variant thereof, under conditions that allow the formation of the trimeric protein complex comprising an active Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof); (b) contacting the trimeric protein complex with a compound and a substrate of the Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof); (c) determining the phosphorylation status of the substrate, wherein the compound is an inhibitor of the trimeric protein complex if the compound inhibits the phosphorylation activity of the Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof).

In some embodiments of the methods of screening for inhibitors of the trimeric protein complexes described herein, the substrate used may comprise a phosphorylation site having the sequence is X₁PX₂X₃(SEQ ID NO: 60), wherein X₁is S or T; X₂is any amino acid; and X₃is K or R. An active Cdk4 (or a variant thereof) or Cdk6 (or a variant thereof) in the complex may phosphorylate the substrate at X₁in the phosphorylation site. In some embodiments, any protein having a phosphorylation site having the sequence is X₁PX₂X₃(SEQ ID NO: 60) may be used in the methods. Examples of a substrate include, but are not limited to, Rb, FoxM1, histone H1, or a variant thereof. The sequences of some exemplary substrates and their variants are listed in Table 4 below.

Methods and techniques for determining the phosphorylation status of a protein are available in the art. For example, radioactive³²P-ATP may be used in phosphorylating a protein.³²P-ATP is subsequently incorporated into the protein. Analysis of the phosphorylated protein may be performed by autoradiography. Other methods for measuring phosphorylation may involve isolating the phosphorylated protein by immunoprecipitation, followed by measurement of reactivity of the phosphorylated protein with a labeled phospho-threonine specific antibody. Antibodies specific for certain phosphorylated threonine residues may also be used directly on live cells with phosphorylated proteins on the cell surface or on whole cell lysates or a mixture of proteins after the lysates or the mixture of proteins are separated by electrophoresis and transferred to a membrane (e.g., PVDF or nitrocellulose in Western blots). Moreover, mass spectrometric techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) may also provide comprehensive parallel analysis of peptide sequences and phosphorylation.

Enzyme-linked immunosorbent assays (ELISAs) may also be used to measure phosphorylation. ELISA may be more quantitative than Western blotting. The format for this microplate-based assay typically utilizes a capture antibody specific for the desired protein, independent of the phosphorylation state in order to first capture the protein on the microplate. A detection antibody specific for the phosphorylation site to be analyzed is then added. These assays are typically designed using colorimetric or fluorometric detection. The intensity of the resulting signal is directly proportional to the concentration of phosphorylated protein present in the original sample. The results from ELISA are easily quantifiable by utilizing a calibrated standard. Further, high specificity is possible due to the use of two antibodies specific for the target protein employed together in the sandwich format. The higher sensitivity often accomplished using ELISAs may allow for smaller sample volumes and the detection of low abundance proteins. Finally, the microplate-based format also allows for much higher throughput than traditional Western blotting.

Human Cdk4 variant (SEQ ID NO: 48), CycD1 variant (SEQ ID NO: 56), and engineered p27 (SEQ ID NO: 6 for amino acids 25-98 of p27 with Y74E) were co-expressed in Sf9 cells (Expression Systems, Davis, Calif.). Cells were simultaneously infected with three baculovirus vectors configured to express the Cdk4 variant, the CycD1 variant, and the engineered p27. Each baculovirus vector was generated using the pFastbac system, which utilizes the polyhedrin promoter. The Cdk4 variant and the engineered p27 were expressed as a GST fusion protein and the CycD1 variant was co-expressed untagged. Lysates were first purified by GS4B affinity chromatography (GE Healthcare). The protein complex was then eluted from the resin and subject to SOURCE 15Q anion exchange chromatography (GE Healthcare). The elution fraction from the anion exchange chromatography was then subjected to TEV protease cleavage overnight in 25 mM Tris, 200 mM NaCl, 1 mM DTT, and 0.5 mM EDTA (pH 8.0) at 4° C. The purified p27-Cdk4-CycD1 trimeric protein complex was then passed over GS4B affinity resin again to remove free GST. The p27-Cdk4-CycD1 trimeric protein complex was then concentrated, and stored in a buffer containing 20 mM Tris, 200 mM NaCl, 1 mM DTT, and 20% glycerol (pH 8.0).

A dimer of Cdk4-CycD1 was first purified following the same protocol of expression and purification as described above, except the baculovirus vector configured to express p27 was left out of the initial infection. Engineered p27 (SEQ ID NO: 6 for amino acids 25-98 of p27 with Y74E) was expressed in E. coli as a fusion protein from a PGEX vector backbone containing T7 promoter. GST-p27 KID fusion was purified as described above.

In order to generate phosphorylated p27 KID, human Brk kinase was expressed in Sf9 cells as a GST fusion protein using the same pFastbac system (polyhedrin promoter). GST-Brk kinase fusion was purified as described above, except the GST fusion tag was not cut. About 100 mg p27 KID was treated with 10% GST-Brk kinase fusion (m/m) in a buffer containing 50 mM Tris, 150 mM NaCl, 1 mM DTT, 10 mM MgCl₂and 1 mM ATP (pH 8.0) and incubated at 4° C. for 24 hours. The phosphorylated p27 was purified by passing through GS4B affinity resin to remove GST-Brk kinase and eluted from a Superdex 75 column (GE Healthcare) in a buffer containing 25 mM Tris, 100 mM NaCl, and 1 mM DTT, (pH 8.0). To form and reconstitute the Cdk4-CycD1-phosp27 trimeric protein complex, three-fold molar excess of phosp27 was mixed with the purified Cdk4-CycD1 dimeric complex. After incubation for 30 minutes on ice, the trimeric protein complex was purified from a Superdex 75 column (GE Healthcare) in a buffer containing 25 mM Tris, 100 mM NaCl, and 1 mM DTT, (pH 8.0).

The phosphorylation activity of Cdk4 in various complexes was tested using different substrates. The protein complexes tested were:

(i) Cdk4-CycD1 dimeric complex (SEQ ID NO: 37 for wild-type Cdk4 and SEQ ID NO: 56 for CycD1 variant),

(ii) Cdk4-CycD1 dimeric complex (SEQ ID NO: 39 for Cdk4 variant having T172E substitution and SEQ ID NO: 56 for CycD1 variant),

(iii) wild-type p27-Cdk4-CycD1 trimeric complex with unphosphorylated p27 (SEQ ID NO: 1 for wild-type p27, SEQ ID NO: 37 for wild-type Cdk4 and SEQ ID NO: 56 for CycD1 variant),

(iv) wild-type p27-Cdk4-CycD1 trimeric complex with unphosphorylated p27 (SEQ ID NO: 1 for wild-type p27, SEQ ID NO: 39 for Cdk4 variant having T172E substitution and SEQ ID NO: 56 for CycD1 variant),

(v) wild-type p27-Cdk4-CycD1 trimeric complex with phosphorylated p27 (SEQ ID NO: 1 for wild-type p27, SEQ ID NO: 37 for wild-type Cdk4 and SEQ ID NO: 56 for CycD1 variant),

(vi) wild-type p27-Cdk4-CycD1 trimeric complex with phosphorylated p27 (SEQ ID NO: 1 for wild-type p27, SEQ ID NO: 37 for Cdk4 variant having T172E substitution and SEQ ID NO: 56 for CycD1 variant),

(vii) engineered p27-Cdk4-CycD1 trimeric complex (SEQ ID NO: 34 or 35 for full-length p27 with Y74E, Y88E, and Y89E, or for amino acids 25-106 of p27 with Y74E, Y88E, and Y89E, respectively, SEQ ID NO: 37 for wild-type Cdk4 and SEQ ID NO: 56 for CycD1 variant), and

(viii) engineered p27-Cdk4-CycD1 trimeric complex (SEQ ID NO: 34 or 35 for full-length p27 with Y74E, Y88E, and Y89E, or for amino acids 25-106 of p27 with Y74E, Y88E, and Y89E, respectively, SEQ ID NO: 37 for Cdk4 variant having T172E substitution and SEQ ID NO: 56 for CycD1 variant).

The substrates used in the kinase assays were the C-terminal domain of the retinoblastoma protein (Rb (771-928); SEQ ID NO: 62), the transactivation domain of FoxM1 (FoxM1 (526-748); SEQ ID NO: 64), and full-length histone H1 (SEQ ID NO: 65).

To observe kinase activity of the Cdk4 or variant thereof in the protein complexes described above, 0.5 μM protein complex was mixed with 20 μM substrate in a buffer containing 25 mM Tris, 200 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 250 μM ATP, and 100 μCi of³²P-γ-ATP (pH 7.0). The substrate was diluted into the reaction buffer at the appropriate concentration, and the reaction was initiated through addition of the complex. The reaction was quenched after 30 minutes through addition of SDS-PAGE loading buffer.

It was found that 1) Cdk4-CycD dimeric complex had high phosphorylation activity specifically for Rb; 2) the wild-type p27-Cdk4-CycD1 trimeric complex with unphosphorylated p27 (e.g., complexes (iii) and (iv) described above) was inhibited; and 3) the wild-type p27-Cdk4-CycD1 trimeric complex with phosphorylated p27 (e.g., complexes (v) and (vi) described above) and the engineered p27-Cdk4-CycD1 trimeric complex (e.g., complexes (vii) and (viii) described above) had phosphorylation activity toward all substrates. Through additional steady state kinetic analysis (FIGS. 1B and 1C), it was found that the engineered p27-Cdk4-CycD1 trimeric complex (e.g., complexes (vii) and (viii) described above) had a reduced KM for ATP. It was also found in a kinase assay that the wild-type p27-Cdk4-CycD1 trimeric complex with phosphorylated p27 and the engineered p27 with Y74E, Y88E, and Y89E-Cdk4-CycD1 trimeric complex were resistant to palbociclib inhibition (FIG. 1D). Compared to the data in FIG. 1D, which used recombinantly expressed proteins in a cell-free system, FIG. 1E shows that endogenous p27-Cdk4-CycD1 trimeric complex immunoprecipitated from cells was also not sensitive to palbociclib inhibition. Indicated cell lysates were immunoprecipitated with control or anti-p27 antibody, and the activity of the immunoprecipitate was used to phosphorylate Rb771-928 with³²P-ATP in the absence or presence of palbociclib. Reactions with the indicated recombinant dimer (K4D1/−) or trimer (K4D1/phosp27) enzymes are shown for comparison in the first four lanes of each SDS-PAGE gel in FIG. 1E. MCF7 and MDA-MB-231 cells are Rb-positive and palbociclib-sensitive breast cancer cells that differ in estrogen receptor status. T98G cells are glioma cells that are relatively less sensitive to palbociclib.

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