Kinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify ...functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of kinase targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options.
Novel-targeted therapies are in rapid development for the treatment of acute lymphoblastic leukemia (ALL) to overcome resistance and decrease toxicity. Survivin, a member of the inhibitor of ...apoptosis gene family and chromosome passenger complex, is critical in a variety of human cancers, including ALL. A well-established suppressor of survivin has been the small molecule, YM155. Reports are identifying other mechanisms of action for YM155. Therefore, we sought to investigate the mode of action and role of YM155 for therapeutic use in the context of ALL.
Primary ALL samples and ALL cell lines were interrogated with YM155 to identify drug sensitivity. Ph(+)ALL harboring the BCR-ABL1 oncogene were tested for any interaction with YM155 and the multi-kinase inhibitor dasatinib. Representative ALL cell lines were tested to identify the response to YM155 using standard biochemical assays as well as RNA expression and phosphorylation arrays.
ALL samples exhibited significant sensitivity to YM155, and an additive response was observed with dasatinib in the setting of Ph(+)ALL. ALL cells were more sensitive to YM155 during S phase during DNA replication. YM155 activates the DNA damage pathway leading to phosphorylation of Chk2 and H2AX. Interestingly, screening of primary patient samples identified unique and exquisite YM155 sensitivity in some but not all ALL specimens.
These results are the first to have screened a large number of primary patient leukemic samples to identify individual variations of response to YM155. Our studies further support that YM155 in ALL induces DNA damage leading to S phase arrest. Finally, only subsets of ALL have exquisite sensitivity to YM155 presumably through both suppression of survivin expression and activation of the DNA damage pathway underscoring its potential for therapeutic development.
The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of ...pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.
Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical ...annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.
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•Acute myeloid leukemia patient cohort with clinical, molecular, drug response data•Validation and discovery of diverse biological features of drug response•Broad mapping of tumor cell differentiation state affecting response to drugs•Modeling reveals a strong and targetable determinant of clinical outcome
Bottomly et al. present a functional genomic resource composed of molecular, clinical, and drug response data on acute myeloid leukemia patient specimens. Through integration of all of these data, they identify genetic and cell differentiation state features that predict drug response, and they utilize modeling to identify targetable determinants of clinical outcome.
There is limited evidence on the optimal strategy for liberating infants and children from invasive mechanical ventilation in the pediatric intensive care unit.
To determine if a sedation and ...ventilator liberation protocol intervention reduces the duration of invasive mechanical ventilation in infants and children anticipated to require prolonged mechanical ventilation.
A pragmatic multicenter, stepped-wedge, cluster randomized clinical trial was conducted that included 17 hospital sites (18 pediatric intensive care units) in the UK sequentially randomized from usual care to the protocol intervention. From February 2018 to October 2019, 8843 critically ill infants and children anticipated to require prolonged mechanical ventilation were recruited. The last date of follow-up was November 11, 2019.
Pediatric intensive care units provided usual care (n = 4155 infants and children) or a sedation and ventilator liberation protocol intervention (n = 4688 infants and children) that consisted of assessment of sedation level, daily screening for readiness to undertake a spontaneous breathing trial, a spontaneous breathing trial to test ventilator liberation potential, and daily rounds to review sedation and readiness screening and set patient-relevant targets.
The primary outcome was the duration of invasive mechanical ventilation from initiation of ventilation until the first successful extubation. The primary estimate of the treatment effect was a hazard ratio (with a 95% CI) adjusted for calendar time and cluster (hospital site) for infants and children anticipated to require prolonged mechanical ventilation.
There were a total of 8843 infants and children (median age, 8 months interquartile range, 1 to 46 months; 42% were female) who completed the trial. There was a significantly shorter median time to successful extubation for the protocol intervention compared with usual care (64.8 hours vs 66.2 hours, respectively; adjusted median difference, -6.1 hours interquartile range, -8.2 to -5.3 hours; adjusted hazard ratio, 1.11 95% CI, 1.02 to 1.20, P = .02). The serious adverse event of hypoxia occurred in 9 (0.2%) infants and children for the protocol intervention vs 11 (0.3%) for usual care; nonvascular device dislodgement occurred in 2 (0.04%) vs 7 (0.1%), respectively.
Among infants and children anticipated to require prolonged mechanical ventilation, a sedation and ventilator liberation protocol intervention compared with usual care resulted in a statistically significant reduction in time to first successful extubation. However, the clinical importance of the effect size is uncertain.
isrctn.org Identifier: ISRCTN16998143.
The optimal first-line mode of noninvasive respiratory support following extubation of critically ill children is not known.
To evaluate the noninferiority of high-flow nasal cannula (HFNC) therapy ...as the first-line mode of noninvasive respiratory support following extubation, compared with continuous positive airway pressure (CPAP), on time to liberation from respiratory support.
This was a pragmatic, multicenter, randomized, noninferiority trial conducted at 22 pediatric intensive care units in the United Kingdom. Six hundred children aged 0 to 15 years clinically assessed to require noninvasive respiratory support within 72 hours of extubation were recruited between August 8, 2019, and May 18, 2020, with last follow-up completed on November 22, 2020.
Patients were randomized 1:1 to start either HFNC at a flow rate based on patient weight (n = 299) or CPAP of 7 to 8 cm H2O (n = 301).
The primary outcome was time from randomization to liberation from respiratory support, defined as the start of a 48-hour period during which the child was free from all forms of respiratory support (invasive or noninvasive), assessed against a noninferiority margin of an adjusted hazard ratio (HR) of 0.75. There were 6 secondary outcomes, including mortality at day 180 and reintubation within 48 hours.
Of the 600 children who were randomized, 553 children (HFNC, 281; CPAP, 272) were included in the primary analysis (median age, 3 months; 241 girls 44%). HFNC failed to meet noninferiority, with a median time to liberation of 50.5 hours (95% CI, 43.0-67.9) vs 42.9 hours (95% CI, 30.5-48.2) for CPAP (adjusted HR, 0.83; 1-sided 97.5% CI, 0.70-∞). Similar results were seen across prespecified subgroups. Of the 6 prespecified secondary outcomes, 5 showed no significant difference, including the rate of reintubation within 48 hours (13.3% for HFNC vs 11.5 % for CPAP). Mortality at day 180 was significantly higher for HFNC (5.6% vs 2.4% for CPAP; adjusted odds ratio, 3.07 95% CI, 1.1-8.8). The most common adverse events were abdominal distension (HFNC: 8/281 2.8% vs CPAP: 7/272 2.6%) and nasal/facial trauma (HFNC: 14/281 5.0% vs CPAP: 15/272 5.5%).
Among critically ill children requiring noninvasive respiratory support following extubation, HFNC compared with CPAP following extubation failed to meet the criterion for noninferiority for time to liberation from respiratory support.
isrctn.org Identifier: ISRCTN60048867.
The optimal first-line mode of noninvasive respiratory support for acutely ill children is not known.
To evaluate the noninferiority of high-flow nasal cannula therapy (HFNC) as the first-line mode ...of noninvasive respiratory support for acute illness, compared with continuous positive airway pressure (CPAP), for time to liberation from all forms of respiratory support.
Pragmatic, multicenter, randomized noninferiority clinical trial conducted in 24 pediatric critical care units in the United Kingdom among 600 acutely ill children aged 0 to 15 years who were clinically assessed to require noninvasive respiratory support, recruited between August 2019 and November 2021, with last follow-up completed in March 2022.
Patients were randomized 1:1 to commence either HFNC at a flow rate based on patient weight (n = 301) or CPAP of 7 to 8 cm H2O (n = 299).
The primary outcome was time from randomization to liberation from respiratory support, defined as the start of a 48-hour period during which a participant was free from all forms of respiratory support (invasive or noninvasive), assessed against a noninferiority margin of an adjusted hazard ratio of 0.75. Seven secondary outcomes were assessed, including mortality at critical care unit discharge, intubation within 48 hours, and use of sedation.
Of the 600 randomized children, consent was not obtained for 5 (HFNC: 1; CPAP: 4) and respiratory support was not started in 22 (HFNC: 5; CPAP: 17); 573 children (HFNC: 295; CPAP: 278) were included in the primary analysis (median age, 9 months; 226 girls 39%). The median time to liberation in the HFNC group was 52.9 hours (95% CI, 46.0-60.9 hours) vs 47.9 hours (95% CI, 40.5-55.7 hours) in the CPAP group (absolute difference, 5.0 hours 95% CI -10.1 to 17.4 hours; adjusted hazard ratio 1.03 1-sided 97.5% CI, 0.86-∞). This met the criterion for noninferiority. Of the 7 prespecified secondary outcomes, 3 were significantly lower in the HFNC group: use of sedation (27.7% vs 37%; adjusted odds ratio, 0.59 95% CI, 0.39-0.88); mean duration of critical care stay (5 days vs 7.4 days; adjusted mean difference, -3 days 95% CI, -5.1 to -1 days); and mean duration of acute hospital stay (13.8 days vs 19.5 days; adjusted mean difference, -7.6 days 95% CI, -13.2 to -1.9 days). The most common adverse event was nasal trauma (HFNC: 6/295 2.0%; CPAP: 18/278 6.5%).
Among acutely ill children clinically assessed to require noninvasive respiratory support in a pediatric critical care unit, HFNC compared with CPAP met the criterion for noninferiority for time to liberation from respiratory support.
ISRCTN.org Identifier: ISRCTN60048867.
We aimed to describe the characteristics and outcome in children with severe
pneumonia in a Chinese PICU.
A retrospective observational study from 2017 to 2019.
A 36-bed university tertiary PICU at ...Shanghai Children's Hospital.
Patients admitted to a tertiary PICU 29 days to 18 years old screened for laboratory-confirmed severe
pneumonia.
None.
Descriptive analysis of baseline characteristics for patients included hospital mortality, organ dysfunctions, use of mechanical ventilation, continuous renal replacement therapy, and/or extracorporeal membrane oxygenation. A total of 817 children with severe pneumonia were admitted to PICU, and 203 of 817 cases (24.8%) with severe
pneumonia were included in this study. The median age was 41 months (interquartile range, 20-67 mo), of which 77.3% (157/203) were younger than 6 years old. Among 163 patients with the test for macrolide resistance, 90.2% cases (147/163) were macrolide-resistant
. Severe
pneumonia-associated organ dysfunction included acute respiratory failure (203 cases, 100%), followed by cardiovascular disorder (79/203, 38.9%), gastrointestinal dysfunction (24/203, 11.8%). The main complications were pleural effusion (79/203, 38.9%), capillary leak syndrome (58/203, 28.6%), and plastic bronchitis (20/203, 9.9%). All patients needed respiratory support, including 64.5% patients (131/203) who received mechanical ventilation and 35.5% patients (72/203) who received high-flow nasal oxygen. Twenty-five patients (12.3%) treated with continuous renal replacement therapy and nine cases (4.4%) received extracorporeal membrane oxygenation. The case fatality rate was 3.9% (8/203). Furthermore, cardiovascular dysfunction, liver injury, or multiple organ dysfunction syndrome were associated with longer mechanical ventilation duration, delayed PICU discharge, and high hospital mortality. Coinfection was a risk factor of delayed PICU discharge.
Children with severe
pneumonia mainly occur under the age of 6 years, showing a high proportion of extrapulmonary organ dysfunction and macrolide resistances. Extrapulmonary organ dysfunction and coinfection are associated with worse outcomes. The overall mortality is relatively low after treated with appreciate antibiotics, respiratory support, and extracorporeal life support.
Despite the impressive success of imatinib in chronic myeloid leukemia (CML), resistance to therapy remains an issue for approximately 15-20% of newly diagnosed chronic phase patients by 5 years and ...transient responses are the rule for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The most frequent and well-characterized mechanism of resistance to imatinib is acquisition of point mutations in the BCR-ABL1 kinase domain which compromise drug binding. This form of resistance is largely well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. However, in the remainder of patients with resistance sustained inhibition of BCR-ABL1 kinase activity is necessary but no longer sufficient to inhibit cell growth, implicating activation of additional, thus far, poorly understood BCR-ABL1-independent mechanisms of growth and survival. In an effort to identify and validate novel pathways important in BCR-ABL-independent resistance to ABL1 kinase inhibitors, we screened a cohort of 30 patients with CML or Ph+ ALL exhibiting clinical resistance to at least one ABL1 kinase inhibitor without evidence of a drug-resistant BCR-ABL1 kinase domain mutation. Following informed consent, fresh primary mononuclear cells were isolated from each sample by Ficoll gradient centrifugation, plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles, and considerable variation in sensitivity profiles for other inhibitors was observed. Among these data, we found a particularly interesting subset of patients whose cells demonstrated ex vivo sensitivity to one or more PI3-K/AKT inhibitors (n=6/30; 20%) including PI-103, BEZ235, CAL-101, and MK-2206. These findings are consistent with previous studies demonstrating PI3-K activation in select ABL1 kinase inhibitor-resistant CML cell lines (Quentmeier et al., J Hem Onc 2011) and in response to imatinib in pre-overt resistance (Burchert et al., Leukemia 2005). Among the effective PI3-K/AKT inhibitors, the most prominent were the dual class IA PI3-K/mTOR inhibitor PI-103 (IC50 range:38-110 nM) and BEZ235 (IC50 range: 20-210 nM). To further interrogate the mechanism(s) behind dependence on this pathway, samples of interest were analyzed by deep sequencing using a custom capture library encompassing ∼2000 kinases, phosphatases, and adaptor proteins. However, in contrast to previous reports of activating PIK3CA mutations in imatinib-resistant KCL-22 cells, we found PIK3CA to be wild-type in all samples. Despite a common sensitivity to PI3-K/AKT pathway inhibition, we found sequence variants were somewhat heterogeneous and direct kinase targets in this pathway were wild-type, suggesting other causative lesions which activate this pathway. Variants were prioritized based on known associations to this pathway, generated and evaluated in vitro for Ba/F3 cell transformation capacity, and validated for pathway activation and kinase inhibitor sensitivity. These results will be presented. Taken together, our findings suggest that a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation demonstrate acquired dependence on the PI3-K/AKT axis, warranting further investigation of inhibitors of this pathway alone and in combination with ABL1 inhibitors as a molecularly targeted therapeutic strategy in patients.
Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Deininger:BMS: Consultancy, Research Funding; ARIAD: advisory board, advisory board Other, Consultancy; Novartis: advisory board, advisory board Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding. Tyner:Incyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, ARIAD & Incyte: Clin trial funding. OHSU holds contracts; Druker receives no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict has been reviewed and managed by OHSU. Other.
Abstract 2481
Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib ...and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs.
Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib.
The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F).
The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells.
No relevant conflicts of interest to declare.
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