Concurrent inhibition of CDK2 adds to the anti-tumour activity of CDK4/6 inhibition in GIST

Abstract

Background: Advanced gastrointestinal stromal tumour (GIST) is characterised by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximising response to therapeutic CDK4/6 inhibition.

Methods: Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples. The impact of inhibitors of CDK2, CDK4 and CDK2/4/6 was determined through cell proliferation and protein detection assays. CDK-inhibitor resistance mechanisms were characterised in GIST cell lines after long-term exposure.

Results: We identify recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation. Moreover, RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) are mechanisms of acquired CDK-inhibitor resistance in GIST.

Conclusions: These studies establish the biological rationale for CDK2 and CDK4/6 co-inhibition as a therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors.

Fig. 1. Frequent dysregulation of the CDK2 and CDK4/6 pathways in clinical samples from metastatic TKI-resistant GIST patients.

a Summary of cell cycle regulator aberrations detected by targeted NGS as well as Immuno-SABER and IHC protein studies in 18 GISTs. Results are shown for low-risk, intermediate-risk, and metastatic stages of GIST progression (risk classifications), and for GISTs initiated by KIT, PDGFRA, NF1, and SDH mutations. Het del: heterozygous deletion; hom del: homozygous deletion. b Targeted NGS reveals co-dysregulation of the CDK2 and CDK4/6 pathways through homozygous co-deletions of CDKN2A/p16, CDKN2A/p14, and/or CDKN2B/p15 coding sequences in five of seven RB1-retained metastatic GISTs. c Immuno-SABER multiplexed and amplified in situ protein imaging demonstrates normal (i.e., retained) expression of key cell cycle markers p16, pRB1 Ser 807/811, RB1, and p53 in representative low-risk (LR; case #1) and intermediate-risk (IR; case #9) GISTs. In contrast, metastatic (Met) GIST #1 (case #12) has a loss of p16 resulting from CDKN2A/p16 homozygous deletion; metastatic GIST #2 (case #13) has p16 overexpression and loss of pRB1 Ser 807/811 and RB1 resulting from RB1 homozygous deletion; and metastatic GIST #3 (case #14) has p16 overexpression and loss of pRB1 Ser 807/811 and RB1 resulting from RB1 homozygous deletion as well as p53 overexpression caused by TP53 missense mutation (p.G245S); retained expression in admixed nonneoplastic cells serves as internal control. Corresponding immunohistochemistry (IHC) (d) and immunoblotting studies (e) validate cell cycle regulator genomic and protein aberrations in GISTs as described above. Positive IHC controls are HPV-associated squamous cell carcinoma (HPV16/18 positive by in situ hybridisation) for p16 and colorectal adenocarcinoma for pRB1 Ser 807/811, RB1 and p53 (d).

Fig. 2. Co-targeting CDK2 and CDK4/6 is synergistic in GIST and inhibits RB1 hyperphosphorylation and cell proliferation.

a Immunoblotting studies showing effects of low-dose palbociclib and low-dose CDK2 inhibitor II single-agent treatment (24 h) on RB1 hyperphosphorylation and cyclin A levels. High-dose palbociclib and CDK2 inhibitor II single-agent treatment (24 h) moderately inhibit pRB1 Ser 795, pRB1 Ser 807/8011 and cyclin A. Effects are maximised when combining low-dose palbociclib and CDK2 inhibitor II. GAPDH serves as loading control and the bar graph normalisations of GIST430 and GIST430/654 are with the higher value set to 100%. b Immunoblotting studies showing effects of treatment (24 h) with the combined CDK2/4/6 inhibitor PF-06873600 on pRB1 Ser 795, pRB1 Ser 807/8011, and cyclin A. Treatment results in inhibition of pRB1 Ser 795, pRB1 Ser 807/8011 and cyclin A along with a slight decrease in total RB1 expression. GAPDH serves as a loading control. Palbociclib (c) and PF-067836000 (d) reduce cell proliferation in GIST430, GIST430/654, and GIST48 as assessed by BrdU incorporation at 24 h (normalised to DMSO). GIST882 (RB1 homozygous deletion) is resistant to palbociclib (c) and shows limited sensitivity to PF-067836000 (d). e Combined inhibition of CDK2 and CDK4/6 using palbociclib and CDK2 inhibitor II was synergistic in GIST430 and GIST430/654, whereas GIST882 was resistant. ** indicates P < 0.01 (one-way ANOVA). Tests were performed in triplicate (mean +/−  s.d). f BrdU incorporation assays highlight the fraction of cells in G1, S, and G2/M phase in GIST430, GIST430/654, GIST48 and GIST882 after treatment with DMSO control (first bar), palbociclib, CDK2 inhibitor II, palbociclib and CDK2 inhibitor II combination and PF-06873600 (1 μM each).

Fig. 3. RB1 inactivation is a mechanism of acquired CDK-inhibitor resistance in GIST.

a Long-term exposure of GIST430/654 to palbociclib causes acquired resistance through RB1 loss as detected by weak RB1 expression at 7 months and complete loss of expression after 11 months by immunoblotting along with loss of pRB1 Ser 795 and a slight increase in cyclin A expression. KIT expression remained stable. GAPDH serves a loading control. b Sanger sequencing at 11 months identifies two new heterozygous RB1 inactivating mutations (c.103C > T; p.Q35*, exon 1; c.2509G > T; p.E837*, exon 24). c Treatment of the resistant GIST430/654/RB1- sub-line with palbociclib (at 31.25, 62.5 and 125 nM), CDK2 inhibitor II (at 500, 1000 and 2000 nM), and combination (palbociclib 31.25 nM + CDK2 inhibitor II 500 nM) has no substantial effect on cyclin A levels. GIST430 serves as RB1-intact control. GAPDH serves as a loading control. A significant shift in palbociclib (d) and PF-06873600 (e) proliferation IC₅₀ values in the palbociclib-resistant GIST430/654/RB1- sub-line compared to parental GIST430/654 cells as assessed by BrdU incorporation at 24 h (normalised to DMSO). ** (7.81 nM) indicates P < 0.01 (one-way ANOVA). Tests were performed in triplicate. The error bars show s.d. f Multiplexed immunofluorescence by Immuno-SABER demonstrates that treatment with palbociclib, the CDK2 inhibitor II, and combination in the RB1-intact GIST430 cell line results in inhibition of RB1 Ser 807/811 and cyclin A expression but has no effect on cyclin A in the resistant GIST430/654/RB1- sub-line with RB1 inactivation.

Fig. 4. Emergence of a novel CCND1 fusion through palbociclib-selective pressure.

a After 14 months of palbociclib treatment, an abnormal form of cyclin D1 (red arrowhead) (termed GIST430/654/D1) emerged in GIST430/654 evidenced by a faint band at 42 kDa, 5 kDa higher than regular cyclin D1 (37 kDa) (black arrowhead); KIT and RB1 expression were preserved; half the amount of protein was loaded for GIST430/654 control because of strong RB1 staining. GAPDH serves as a loading control. b Immunoprecipitation studies demonstrate detection of the abnormal 42-kDa cyclin D1 band (red arrowhead) in input and cyclin D1 IP lanes in the palbociclib-resistant sub-line termed GIST430/654/D1 by an antibody directed against full-length cyclin D1 (sc-20044), but not by those directed against the C-terminus (sc-718) or pCyclin D1 Thr 286. Background levels of wild-type cyclin D1 (black arrowheads) are detected by the full-length, C-terminal and pCyclin D1 Thr 286 antibodies in this cell line and in input and cyclin D1 IP lanes in GIST-T1 which has weak levels of cyclin D1 and serves as control. Co-IP detects interaction of the 42-kDa cyclin D1 protein with CDK4 in GIST430/654/D1. Beads-only lanes are negative control. c Mass spectrometry on cyclin D1 IP bands isolated from a Coomassie-stained gel with and without iodoacetamide (IA) treatment. Alkylation improved separation on SDS-PAGE and facilitated mass spectrometry analysis. Analysis of the isolated 42-kDa band (red) identifies a novel cyclin D1 peptide sequence after amino acid (AA) 279 (green). Analysis of the isolated 37-kDa cyclin D1 band (blue) reveals wild-type peptides (green). Parental GIST430/654 with weak expression of wild-type cyclin D1 serves as a reference. Beads-only lanes are negative controls. d 3’ rapid amplification of cDNA ends (RACE) followed by nested polymerase chain reaction (PCR) and Sanger sequencing identifies the presence of an intragenic rearrangement between CCND1 exon 5 and the 3’ flanking sequence of AP003555.2, a long non-coding RNA located ~300 kb 3’ of CCND1. e RNA sequencing confirms the breakpoints in CCND1 and the 3’ flanking sequence of AP003555.2 (red arrows). f Whole-exome sequencing detects the CCND1::chr11.g:70025223 rearrangement (breakpoints indicated by red arrows). A significant shift in palbociclib (g) and PF-06873600 (h) proliferation IC₅₀ values in the palbociclib-resistant GIST430/654/D1 sub-line compared to parental GIST430/654 cells as assessed by BrdU incorporation at 24 h (normalised to DMSO). ** (7.81 nM) indicates P < 0.01 (one-way ANOVA). Tests were performed in triplicate. The error bars show s.d.

Fig. 5. The novel oncogenic form of cyclin D1 decreases CDK4/6 inhibitor sensitivity when overexpressed in cell lines.

a Schematic of the fusion between CCND1 exon 5 and the 3’ flanking sequence of AP003555.2, which results in a novel oncogenic form of cyclin D1. The 3’ flanking sequence of AP003555.2 is inserted in the opposite (i.e., 3’–5’ instead of 5’–3’) read direction (top). Replacement of the cyclin D1 C-terminus by the novel AA sequence results in loss of Thr 286 and Thr 288, two residues essential for cyclin D1 phosphorylation and proteasomal degradation (bottom). b Treatment with palbociclib (100 nM) from day 6–13 post infection results in growth inhibition of GIST430/654 cells infected with pLKO.1 empty vector but has no effect on the GIST430/654 cells infected with the CCND1 fusion construct. c Expression of the CCND1 fusion construct in the RB1-intact GIST430/654, LMS05 and BT474 cell lines at 4, 10 and 10 days post infection, respectively. No expression of cyclin D1 is detected in the untreated and empty vector controls. GAPDH serves as a loading control. d Treatment of GIST430/654 with palbociclib for 26 days further increases expression of the CCND1::chr11.g:70025223 construct and mildly decreases expression of pRB1 Ser 795. Total RB1 expression is unchanged. Shown are regular (1 min) and short (15 sec) exposures for cyclin D1. GAPDH serves as a loading control. e Decreased sensitivity to palbociclib in GIST430/654, LMS05 and BT474 infected with the CCND1::chr11.g:70025223 construct compared to empty vector-infected cells as assessed by BrdU incorporation at 24 h (normalised to DMSO). * indicates P < 0.05; ** indicates P < 0.01 (one-way ANOVA). Tests were performed in triplicate. The error bars show s.d.

Fig. 6. RNA sequencing confirms that RB1 inactivation and a novel oncogenic form of cyclin D1 confer palbociclib resistance.

a Unsupervised hierarchical clustering demonstrates that DMSO-control and palbociclib-treated conditions cluster together within GIST430/654 parental (parental), the sub-line with RB1 inactivation (RB1-), and the sub-line with CCND1 fusion. b Gene expression results by FKPM show near-absence of cyclin D1 expression in GIST430/654 parental (parental), low levels in the sub-line with RB1 inactivation (RB1-), and very high levels in the sub-line with CCND1 fusion. RB1 expression is detected at moderate levels in GIST430/654 parental and the sub-line with CCND1 fusion and is very low in the sub-line with RB1 inactivation. c Gene ontology (GO) analysis in GIST430/654 parental after treatment with palbociclib detects enrichment of cell cycle/growth-associated GO terms, specifically, cell division, cell proliferation, mitotic nuclear division, sister chromatid segregation, and chromosome segregation GO terms (top). The number of significant genes per category are shown at the bottom. No enrichment of GO terms is detected in the sub-lines with RB1 inactivation and the CCND1 fusion. d Volcano plots demonstrate a number of significantly differentially expressed genes GIST430/654 parental and none in those with RB1 inactivation and the CCND1 fusion confirming the presence of palbociclib resistance on the RNA transcriptomic level.