Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor

Abstract

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

Figures

Fig 1. CT scan of index patient

CT scan showing a liver lesion experiencing a partial response after 6 cycles of BYL719

Fig. 2. Gene copy number variation in both primary tumour and lung metastasis

Fig. 3. Representative exon-level copy number profiles for genes on chromosome 10

Exons in PTEN are shown in red.

Fig. 4. Loss-of-function mutations in PTEN detected by MSK-IMPACT

Mutations were visualized by the Integrative Genomics Viewer (IGV).

Fig 5. PTEN immunostaining of the 14 metastases collected during the autopsy

Hematoxylin and eosin (H&E) and PTEN expression detected by IHC in 14 metastases collected during the autopsy of the index patient. PTEN staining in PTEN negative samples is only present in stromal cells.

Fig 6. PTEN immunostaining in patients treated with BYL719

PTEN expression detected by IHC in paired samples from six additional patients treated with BYL719. Specimens before starting BYL719 therapy (baseline) and at time of disease progression (post-treatment) are compared.

Fig 7. Inducible loss of PTEN and sensitivity to BYL719 and BKM120

a. Cell viability assay in MCF7 cells with inducible PTEN knockdown treated with increasing concentrations of either BYL719 or BKM120. Error bars indicate SEM. b. Cell viability assay in MDA-MB-453 (MDA453) cells with constitutive PTEN knockdown treated with increasing concentrations of either BYL719 or BKM120. Error bars indicate SEM. c. Quantification of pAKT (S473) and pS6 (S240/4) from Figure 4d. Student t-test was used and p values are indicated. d. Western blot from the PDXs treated as indicated.

Fig 8. Constitutive loss of PTEN and sensitivity to BYL719 and AZD6482

a. Cell viability assay in T47D cells with inducible PTEN knockdown (#2) treated with increasing concentrations of either BYL719 or AZD6482 in the presence of doxycycline 1μg/mL. Error bars indicate SEM. b. Quantification of pAKT (S473) and pS6 (S240/4) from Figure 4g. Student t-test was used and p values are indicated. Error bars indicate SEM. c. Western blot from the PDXs treated as indicated.

Figure 1. Clinical response of index patient treated with BYL719

CT scans showing stable peri-aortic lymph node metastasis (yellow circle) and the appearance of new lung metastases (yellow circles) after the completion of the tenth cycle of BYL719 therapy. Arrow: pleural effusion.

Figure 2. Loss of PTEN upon BYL719 resistance

a, Circos plots from WGS analysis of primary tumour (prior to BYL719 treatment) and a lung metastasis appearing after the tenth cycle of BYL719 therapy. b, CNV of chromosome 10. c, WES of the peri-aortic lymph node showing durable stable disease during BYL719 therapy compared to both primary tumour and the progressing lung lesion. The diagram shows the variation of allele frequencies (VAF) of the listed gene mutations in the three lesions. The estimated tumour purities are 44% for the breast primary tumour, 50% for the lung metastasis, and 59% for the lymph node metastasis. d, PTEN IHC of primary tumour, peri-aortic lymph node, and lung metastasis. Images were taken from Servier Medical Art.

Figure 3. Loss of PTEN by different genetic alterations

a, Heatmap of the non-silent genetic alterations across the primary tumour and the 14 metastases collected during the autopsy of the index patient. Gene mutations are depicted in green, gene amplifications in red, and gene copy number loss in blue. b, Dendrogram showing the proposed phylogenetic evolution of the metastases in the index patient. Shadowed circles represent metastatic lesions with bi-allelic loss of PTEN and lack of PTEN expression by IHC.

Figure 4. Loss of PTEN expression and sensitivity to PI3Kα and PI3Kβ blockade

a, Western blot showing PTEN knockdown by two independent shRNAs and its effects on the PI3K/AKT/mTOR pathway. b, Cell viability assay in T47D cells with inducible PTEN knockdown treated with increasing concentrations of BYL719 or BKM120. Error bars represent standard error of the mean (SEM). c, Antitumour activity of either BYL719 (25mg/kg daily) or BKM120 (25mg/kg daily) in PDXs subcutaneously grown in nude mice (n=6 (Vehicle) and n=8 (Treatments)). Error bars represent SEM. d, Representative immunostaining for phosphorylated AKT (pAKT) and phosphorylated S6 (pS6) in PDXs treated as shown. Tumours were collected at the end of the experiment of Panel c, 2 hours after the last dosage. Scale bar represents 100μm. e, Cell viability assay in T47D cells with PTEN expression (shRenilla) or PTEN knockdown (shPTEN#2) treated with increasing concentrations of the combination of BYL719 and AZD6482. Error bars represent SEM. f, Antitumour activity of either BYL719 (25mg/kg daily) or the combination of BYL719 and AZD6482 (25mg/kg daily) in PDXs subcutaneously grown in nude mice (n=6 (Vehicle) and n=8 (Treatments)). Error bars represent SEM. g, Representative immunostaining for phosphorylated AKT (pAKT) and phosphorylated S6 (pS6) in PDXs treated as shown. Tumours were collected at the end of the experiment of panel f, 2 hours after the last dosage. Scale bar represents 100μm. * p<0.05.