BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

The PARP1 selective inhibitor saruparib (AZD5305) elicits potent and durable antitumor activity in patient-derived BRCA1/2-associated cancer models.

BACKGROUND: Poly (ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors (PARPi) are targeted therapies approved for homologous recombination repair (HRR)-deficient breast, ovarian, pancreatic, and prostate cancers. Since inhibition of PARP1 is sufficient to cause synthetic lethality in tumors with homologous recombination deficiency (HRD), PARP1 selective inhibitors such as saruparib (AZD5305) are being developed. It is expected that selective PARP1 inhibition leads to a safer profile that facilitates its combination with other DNA damage repair inhibitors. Here, we aimed to characterize the antitumor activity of AZD5305 in patient-derived preclinical models compared to the first-generation PARP1/2 inhibitor olaparib and to identify mechanisms of resistance. METHODS: Thirteen previously characterized patient-derived tumor xenograft (PDX) models from breast, ovarian, and pancreatic cancer patients harboring germline pathogenic alterations in BRCA1, BRCA2, or PALB2 were used to evaluate the efficacy of AZD5305 alone or in combination with carboplatin or an ataxia telangiectasia and Rad3 related (ATR) inhibitor (ceralasertib) and compared it to the first-generation PARPi olaparib. We performed DNA and RNA sequencing as well as protein-based assays to identify mechanisms of acquired resistance to either PARPi. RESULTS: AZD5305 showed superior antitumor activity than the first-generation PARPi in terms of preclinical complete response rate (75% vs. 37%). The median preclinical progression-free survival was significantly longer in the AZD5305-treated group compared to the olaparib-treated group (> 386 days vs. 90 days). Mechanistically, AZD5305 induced more replication stress and genomic instability than the PARP1/2 inhibitor olaparib in PARPi-sensitive tumors. All tumors at progression with either PARPi (39/39) showed increase of HRR functionality by RAD51 foci formation. The most prevalent resistance mechanisms identified were the acquisition of reversion mutations in BRCA1/BRCA2 and the accumulation of hypomorphic BRCA1. AZD5305 did not sensitize PDXs with acquired resistance to olaparib but elicited profound and durable responses when combined with carboplatin or ceralasertib in 3/6 and 5/5 models, respectively. CONCLUSIONS: Collectively, these results show that the novel PARP1 selective inhibitor AZD5305 yields a potent antitumor response in PDX models with HRD and delays PARPi resistance alone or in combination with carboplatin or ceralasertib, which supports its use in the clinic as a new therapeutic option.

The ATR inhibitor ceralasertib potentiates cancer checkpoint immunotherapy by regulating the tumor microenvironment.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Relevance of ATM Status in Driving Sensitivity to DNA Damage Response Inhibitors in Patient-Derived Xenograft Models.

Ataxia-telangiectasia mutated gene (ATM) is a key component of the DNA damage response (DDR) and double-strand break repair pathway. The functional loss of ATM (ATM deficiency) is hypothesised to enhance sensitivity to DDR inhibitors (DDRi). Whole-exome sequencing (WES), immunohistochemistry (IHC), and Western blotting (WB) were used to characterise the baseline ATM status across a panel of ATM mutated patient-derived xenograft (PDX) models from a range of tumour types. Antitumour efficacy was assessed with poly(ADP-ribose)polymerase (PARP, olaparib), ataxia- telangiectasia and rad3-related protein (ATR, AZD6738), and DNA-dependent protein kinase (DNA-PK, AZD7648) inhibitors as a monotherapy or in combination to associate responses with ATM status. Biallelic truncation/frameshift ATM mutations were linked to ATM protein loss while monoallelic or missense mutations, including the clinically relevant recurrent R3008H mutation, did not confer ATM protein loss by IHC. DDRi agents showed a mixed response across the PDX's but with a general trend toward greater activity, particularly in combination in models with biallelic ATM mutation and protein loss. A PDX with an ATM splice-site mutation, 2127T > C, with a high relative baseline ATM expression and KAP1 phosphorylation responded to all DDRi treatments. These data highlight the heterogeneity and complexity in describing targetable ATM-deficiencies and the fact that current patient selection biomarker methods remain imperfect; although, complete ATM loss was best able to enrich for DDRi sensitivity.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models.

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

The relationship between transitional motor skills and locomotion.

This study explores whether transitional skills and sitting correlate with locomotion onset. The development of eight infants was followed. Most transitional skills correlated with locomotor skills. Sitting and rolling did not. Transitional skills may resemble the control needed for locomotion more closely than sitting.