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.
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 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.
BackgroundThe combination of an ATR inhibitor ceralasertib and anti-PD-L1 antibody durvalumab is being tested in Phase III clinical trials in patients who have progressed on prior immunotherapy. ...Preclinical experiments were performed to build a greater understanding of the potential immune driven mechanisms-of-action by which ceralasertib enhances antitumor efficacy in combination with anti-PD-L1 in the context of the clinical dose and schedule.MethodsTo assess the antitumor efficacy and associated pharmacodynamic effects ceralasertib ceralasertib was administered to CT26 tumor-bearing BALB/c immunocompetent mice twice daily with continuously and intermittently 7 day-on/7 day-off schedules alone or in combination with PD-L1 blockade. Flow cytometry and immunohistochemistry was used to quantify CD8+ T-cells when on and off ceralasertib treatment. Immunophenotyping was performed using single-cell CyTOF protein expression mass cytometry and bulk tumor or single-cell RNA sequencing (scRNA-seq) transcriptomics analysis to evaluate intratumoral T-cell populations.ResultsModelling of intermittent ceralasertib dosing regimen in mouse tumor models revealed CD8+ T-cell and type I interferon (IFNI) dependent antitumor activity, which was enhanced in combination with anti-PD-L1 immune checkpoint blockade. Ceralasertib suppressed highly proliferating CD8+ T-cells when on-treatment which was rapidly reversed when off-treatment. This was linked to significant relative reductions in T-cells which displayed markers commonly associated with an exhausted phenotype and a concomitant increase in cells with naïve non-activated phenotypes with improved T-cell function in the tumor microenvironment. Continuous daily administration of ceralasertib led to the sustained suppression of CD8+ T-cells and impaired antitumor activity compared to intermittent dosing. In addition, ceralasertib caused up-regulation of type I interferon (IFNI) which may promote an inflammatory environment and drive direct anti-proliferative effects on tumor cells.ConclusionsBroad immunostimulatory and immunomodulatory changes following intermittent ATR treatment in combination with immune checkpoint blockade may underpin the durable clinical therapeutic benefit observed in patients and indicates its potential wider impact on antitumor immunity.
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.
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.
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.
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.
•This study explores how sitting and transitional motor skills relate to walking attainment.•Independent sitting when placed was not significantly correlated to the attainment of walking.•The many ...transitional motor skills correlated with walking.•The pelvic and trunk control associated with sitting alone may be insufficient for locomotion.•The pelvic and trunk control needed for walking may be gained by practicing transitional skills.
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.
