Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2

Abstract

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.

Variants of uncertain significance in the era of high-throughput genome sequencing: a lesson from breast and ovary cancers

“…Variant experimental analysis has led to in vitro treatments with specific agents that have been progressively introduced in the clinical practice. An example are the poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), which were demonstrated to be effective in breast and ovarian cancers bearing mutations not only in BRCA1 and BRCA2, but also in genes involved in the DNA damage homologous recombination pathway, such as PALB2, RAD51, ATM, ATR and CHK2 [52,53]. The observation that also other "BRCAness" genes were involved in PARPi sensitivity opened the possibility of investigating other kinds of malignancies with dysfunction in the DNA damage response, where these genes are high-to-intermediate risk factors [54].…”

“…They range from homology-directed DNA repair assays [55,56], to in vitro transactivation of specific domains and measurements of ubiquitin ligase activity [57]. Studies with conditional knock-out cells allow to evaluate the capacities of HBOC-related gene variants to rescue lethality, DNA repair, and resistance to PARPi [48,53,58,59]. Other assays focus on micronucleus formation and centrosome amplification in mutated cell lines [60,61], on the restoration of resistance to damaging agents by BRCA2 re- introduction [60], or on increased chromosome breakage after γ-irradiation [62].…”

Molecular Mechanisms of PALB2 Function and Its Role in Breast Cancer Management

“…Moreover, p.P8L (c.23C>T), p.K18R (c.53A>G), p.L24S (c.71T>C), p.Y28C (c.83A>G), and p.R37H (c.110G>A) compromise HR activity of PALB2 and are suggested to be pathogenic (106)(107)(108). In the PALB2 C-terminus, p.W912G, p.G937R, p.L939W, p.I944N (c.2831T>A), p.L947F (c.2841G>T), p.L947S (c.2840T>C), p.L961P, p.L972Q, p.A1025R, p.T1030I (c.3089C>T), p.I1037T, p.G1043D, p.L1070P (c.3209T>C), p.P1088S (c.3262C>T), p.W1140G (c.3418T>C), p.L1143P, and p.L1172P promoted a decrease in the HR activity of PALB2 (20,(107)(108)(109)112). Among these mutations, p.L939W, p.A1025R, p.T1030I, p.P1088S, and p.L1143P disrupt BRCA2-PALB2 interaction; p.W912G, p.G937R, p.I944N, p.L961P, p.L972Q, p.T1030I, p.I1037T, p.G1043D, and p.L1172P are associated with PALB2 protein instability; and p.I944N, p.L947F, p.L947S, p.T1030I, p.L1070P, and p.W1140G result in the mislocalization of PALB2 to the cytoplasm.…”

“…A concurrent study conducted by Wiltshire et al revealed four new PARPi-hypersensitive variants in PALB2 (p.L24S, p.I944N, p.A1025R, and p.L1070P) using PALB2-deficient B400 mouse mammary tumor cells (108). Boonen et al(109) developed a cDNA-based system for the functional analysis of PALB2 variants. By evaluating the ability of PALB2 variants to rescue PARPi sensitivity in PALB2 knockout mouse embryonic stem cells, they identified twelve PALB2 variants (p.Y28C, p.L35P, p.W912G, p.G937R, p.I944N, p.L947S, p.L961P, p.L972Q, p.A1025R, p.T1030I, p.G1043D, and p.L1172P) that showed hypersensitivity to PARPi(109).In spite of the lack of clinical evidence for PARPi treatment efficacy in PALB2-deficient breast cancer patients, the response of some other PALB2-deficient solid tumors to PARPi in clinical/preclinical studies have been remarkable.…”

“…Boonen et al(109) developed a cDNA-based system for the functional analysis of PALB2 variants. By evaluating the ability of PALB2 variants to rescue PARPi sensitivity in PALB2 knockout mouse embryonic stem cells, they identified twelve PALB2 variants (p.Y28C, p.L35P, p.W912G, p.G937R, p.I944N, p.L947S, p.L961P, p.L972Q, p.A1025R, p.T1030I, p.G1043D, and p.L1172P) that showed hypersensitivity to PARPi(109).In spite of the lack of clinical evidence for PARPi treatment efficacy in PALB2-deficient breast cancer patients, the response of some other PALB2-deficient solid tumors to PARPi in clinical/preclinical studies have been remarkable. A preclinical study of BMN673 (talazoparib) for the panel of the Pediatric Preclinical Testing Program showed that a maintained complete response was observed in vivo in a Wilms tumor xenograft model, characterized by a truncating mutation in PALB2 (p.Y1108fs)(110).…”