Hypoxic tumor cells are likely to be resistant to conventional chemotherapy, in large part because many anticancer drugs are unable to penetrate into poorly oxygenated tumor tissue. Here, we used ...quantitative immunofluorescence to study the distribution of mitoxantrone and AQ4N in tumor tissue. AQ4N is a prodrug activated under hypoxic conditions to AQ4, which is structurally similar to mitoxantrone and inhibits topoisomerase II. We characterized the penetration of mitoxantrone and AQ4N/AQ4 through multilayered cell cultures (MCC) and in relation to blood vessels and hypoxic regions in human tumor xenografts. We also studied tumor growth delay after treatment with each agent alone and with the combination. In both MCC and xenografts, mitoxantrone is taken up by proximal cells and penetrates slowly to distant regions. In contrast, AQ4N rapidly penetrates MCC and tumor tissue in vivo, and AQ4N (or its reduced form AQ4) is detected at high concentration within hypoxic regions. The targeting of mitoxantrone to oxygenated regions and AQ4N/AQ4 to hypoxic tumor regions results in effective drug exposure over the entire tumor after combined treatment and increases tumor growth delay compared with either drug alone. The combination of a clinically used anticancer drug with limited tissue penetration and a structurally related drug activated in regions of tumor hypoxia is an effective strategy to overcome chemoresistance due to the tumor microenvironment. This study supports clinical evaluation of AQ4N in combination with conventional anticancer agents, such as mitoxantrone.
Purpose: AQ4N is a novel prodrug that is selectively bioreduced to AQ4, a topoisomerase II inhibitor, in hypoxic tumor. This study
assessed the maximum tolerated dose and pharmacokinetics of AQ4N ...when administered weekly in patients with advanced cancers.
Experimental Design: AQ4N was administered as a 30-minute i.v. infusion on days 1, 8, and 15 of a 28-day cycle in eight dose cohorts ranging from
12 to 1,200 mg/m 2 . Accelerated titration design was used and the maximum tolerated dose was defined as the highest dose at which fewer than
two of six patients had a dose-limiting toxicity.
Results: Sixteen patients were treated with cumulative doses of AQ4N ranging from 61.6 through 9,099.1 mg/m 2 . A single patient per cohort was treated up to 384 mg/m 2 without toxicities. At 1,200 mg/m 2 , two of five patients experienced a dose-limiting toxicity (grade 5 respiratory failure and grade 3 fatigue). Five cohort
assigned patients were treated without toxicity at 768 mg/m 2 , establishing this dose as the maximum tolerated dose. Among the most common adverse events observed were fatigue (38%),
diarrhea (31%), nausea (25%), vomiting (25%), and anorexia (13%). Anticipated blue coloration of body fluids or skin was observed
in all patients. The pharmacokinetics of AQ4N were dose proportional over all doses studied. Three patients experienced stable
disease, including a patient with collecting duct renal cancer stable for 25 months.
Conclusion: AQ4N is well tolerated when administered weekly on a 3-of-4-week schedule at 768 mg/m 2 . Further combination studies investigating the safety and efficacy of AQ4N are ongoing.
The 'Competing interests' statement of this Article has been updated; please see the accompanying Amendment. The original Article has not been corrected online.
We report a
gatekeeper mutation in a patient with
-mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that
is a neratinib-sensitive, gain-of-function mutation ...that upon dimerization with mutant HER3
, also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression,
was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2
reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2
but not HER2
In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2
-induced signaling and cell growth. Acquisition of HER2
upon development of resistance to neratinib in a breast cancer with an initial activating
mutation suggests
is a driver mutation. HER2
-mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib.
We found an acquired
gatekeeper mutation in a patient with
-mutant breast cancer upon clinical progression on neratinib. We speculate that
may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with
-activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib.
.
Use of Chemokine Receptors by Poxviruses Lalani, Alshad S.; Masters, Jennefer; Zeng, Wei ...
Science (American Association for the Advancement of Science),
12/1999, Volume:
286, Issue:
5446
Journal Article
Peer reviewed
Chemokine receptors serve as portals of entry for certain intracellular pathogens, most notably human immunodeficiency virus (HIV). Myxoma virus is a member of the poxvirus family that induces a ...lethal systemic disease in rabbits, but no poxvirus receptor has ever been defined. Rodent fibroblasts (3T3) that cannot be infected with myxoma virus could be made fully permissive for myxoma virus infection by expression of any one of several human chemokine receptors, including CCR1, CCR5, and CXCR4. Conversely, infection of 3T3-CCR5 cells can be inhibited by RANTES, anti-CCR5 polyclonal antibody, or herbimycin A but not by monoclonal antibodies that block HIV-1 infection or by pertussis toxin. These findings suggest that poxviruses, like HIV, are able to use chemokine receptors to infect specific cell subtypes, notably migratory leukocytes, but that their mechanisms of receptor interactions are distinct.
Abstract
Crofelemer is a novel allosteric modulator of cystic fibrosis transmembrane conductance regulator (CFTR) that is approved as an antidiarrheal in HIV patients receiving antiretroviral ...therapy. Neratinib is an oral, irreversible pan-HER tyrosine kinase inhibitor approved for extended adjuvant treatment of early stage HER2+ breast cancer. The objective of this study was to evaluate the effects of crofelemer in reducing the incidence and severity of diarrhea following daily oral neratinib dosing for 28 days in healthy female dogs, without concomitant use of loperamide.
Female beagle dogs (mean 7.2 kg (6.4-8.5)) were randomized into three groups of 8 dogs each and all groups received neratinib oral doses (40 mg for the first 5 days, then 80 mg) for 28 days. Group 1 dogs received one placebo capsule orally four times daily, control (CTR) group; group 2 dogs received one crofelemer 125 mg tablet four times a day (QID group); and group 3 dogs received one crofelemer 125 mg tablet twice daily (BID group). Dogs were evaluated twice daily for bowel movements, which were scored according to a 7-point scale analogous to the human Bristol Stool Form Scale. Dogs with moderate dehydration were given subcutaneous fluids and/or a single-day neratinib dose reduction or holiday. Weekly assessments of clinical chemistry and hematology parameters were conducted. Pharmacological effects were assessed by: 1) determining the proportion of “responder” dogs, defined as dogs with <7 watery stools per week for at least 2 weeks of the 4 week period; 2) reduction in the number of watery stools over the 28-day period; and 3) assessment of change in stool consistency. Summary statistics were computed at each week and pair-wise p-values were computed via t-test to determine differences from control group. Analysis of covariance (ANCOVA) was conducted using baseline fecal scores as a covariate.
Following 28 days of oral dosing of neratinib receiving concomitant crofelemer tablets or placebo capsules, 3 of 8 placebo (CTR) dogs were “responders”; 6/8 crofelemer BID group dogs (p=0.03) and 7/8 QID group dogs (p=0.02) were “responders”. The average number of watery stools per week, a measure of the incidence of diarrhea, across the 4-week treatment period were 9, 6, and 6 for the control, BID, and QID groups (p=0.01) respectively. The average number of weeks with no loose/watery stools were determined to be 1.3, 2.1, and 2.3 for the control, BID, and QID groups respectively, with 1-sided p-values of 0.043 and 0.0295, respectively. Least squares weekly mean fecal scores, a measure of diarrhea severity reflecting stool consistency, averaged across the 4-week period were 5.1, 3.9, and 4.1 for control, BID, and QID groups (p=0.005 and p=0.017, respectively).
In this experimental preclinical model, concomitant treatment of crofelemer without any use of loperamide reduces the incidence and severity of neratinib-associated diarrhea in female dogs by about 30% over a 28-day treatment period.
Citation Format: Michael K. Guy, Andre Teixeira, Alshad S. Lalani, Irmina Diala, Leanne McCulloch, James Bolognese, Pravin Chaturvedi. Effects of oral crofelemer on neratinib-induced diarrhea in beagle dogs abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 580.
Abstract
Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have ...indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers.
We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance.
Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1).
Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W
Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2420.
Abstract
Background: We previously showed preclinical and clinical activity of combined inhibition of EGFR and PARP with lapatinib and veliparib in triple negative breast cancer (Nowsheen et al. PLoS ...One 2012;7(10):e46614; Reasor et al. J Clin Onc 2018; 36(15_suppl):1095), due to an induced homologous recombination repair defect with EGFR inhibition sensitizing cells to PARP inhibition. In this study, we tested the in vitro and in vivo activity of the irreversible pan-HER tyrosine kinase inhibitor, neratinib, with the PARP inhibitor olaparib or niraparib.
Methods: Human triple negative breast cancer (TNBC) cells MDA-MB231 (BRCA wt), MDA-MB453 (BRCA wt), and MDA-MB436 (BRCA1 mutant) and Mouse TNBC 4T1 cells (BRCA wt) were used. Cell proliferation and colony formation assays were performed to assess the in vitro activity of various concentrations of neratinib and olaparib. Markers of DNA damage were measured via gamma-H2AX western blot analysis. In vivo effects of combination treatments on tumor metastasis were tested using intracardiac injection of tumor cells labelled with luciferase and mice were monitored for survival.
Results: Combining neratinib with olaparib or niraparib significantly decreased proliferation compared to either agent alone in MDA-MB231, MDA-MB453, MDA-MB436 and 4T1 cells. Similar results were observed using colony formation assays. Cytotoxicity was associated with increased gamma-H2AX levels, indicating persistent DNA damage. Mice treated with 20mg/kg/day of neratinib and 100mg/kg/day of olaparib after MDA-MB231-Luc intracardiac injection exhibited reduced tumor metastases compared to either agent alone, resulting in better survival (p=0.0088).
Conclusions: Combining neratinib with PARP inhibition induces synthetic lethality resulting in increased anti-tumor activity in TNBC in cell lines and in xenografts. Future clinical trials testing these combinations are warranted.
Citation Format: Chuan Xing, Zhuo Zhang, Deborah Della Manna, Irmina Diala, Lisa Eli, Alshad S. Lalani, Eddy Shih-Hsin Yang. Neratinib induces synthetic lethality with PARP inhibitors in triple negative breast cancer cells in vitro and in vivo abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1181.
Abstract
Introduction
Neratinib is an irreversible pan-HER tyrosine kinase inhibitor used for treatment of HER2+ breast cancer. Diarrhea is a common toxicity observed with this class of agents. We ...have previously shown that neratinib-induced diarrhea is associated with alterations to the gut microbiome in a rat model Secombe et al. 2020. This study aimed to evaluate contribution of specific gut microbiota in the development of neratinib-induced diarrhea using different classes of oral antibiotics.
Methods
Female Albino Wistar rats (n=44) were treated daily with 50 mg/kg neratinib or vehicle control (0.5% hydroxypropyl methylcellulose) for 28 days by oral gavage. Antibiotics (vancomycin (0.5g/L), neomycin (1g/L) or an antibiotic cocktail (vancomycin 0.5g/L, neomycin 1g/L and ampicillin 1g/L)) were administered via drinking water throughout the study, beginning four weeks prior to neratinib. Diarrhea was scored from 0-3. In the vehicle control, neratinib alone and neomycin treated groups, fecal contents were collected at necropsy and subject to 16S bacterial DNA sequencing and bioinformatic analysis was performed to characterize the microbiome.
Results
Conclusions
Neratinib treatment was associated with an increase in Gram negative Proteobacteria, and neomycin targeting Gram negative bacteria was most effective at reducing neratinib-induced diarrhea. These studies suggest that modulating the microbiome may be a novel strategy for mitigating or prevention of tyrosine kinase inhibitor-induced diarrhea. Given that increases in Blautia were associated with ablation of diarrhea, this is now a sensible further target for future intervention studies.
VariableVehicle Control(n=8)Neratinib(n=10)Neratinib + antibiotic cocktail (n=8)Neratinib + vancomycin (n=8)Neratinib + neomycin(n=10)Incidence of diarrhea, %Grade 010014.112.192.295.2Grade 1026.278.46.94.8Grade 2047.67.80.80Grade 3012.1000Mean±SEM days with grade 2 diarrhea0±0*13.8±1.592.25±0.620.25±0.16*0±0*Median (range) tissue injury scoreProximal ileum0.5 (0-2)2 (1-4)**1.5 (0-4)1.5 (0-4)2 (1-4)Distal ileum1 (0-2)3 (1-4)**3 (0-4)3 (2-4)**3 (2-4)**Proximal colon1 (0-2)2 (0-3)1.5 (1-3)1.5 (0-2)1 (0-2)Distal colon1 (0-2)3 (1-4)2 (1-3)2 (1-3)2 (1-3)Microbial analysisShannon's Index (mean±SEM)2.42±0.17#2.39±0.15#--1.30±0.11Relative genus abundanceProteobacteria0.0076±0.0010.045±0.01#0.0045±0.001Blautia0.042±0.01#0.016±0.005#--0.70±0.04Allobaculum0.0045±0.0020.17±0.04**--0.074±0.03SEM, standard error of the mean, PCoA, Principal Coordinate Analysis*P<0.005 vs neratinib, **P<0.05 vs control, #P<0.005 vs neratinib +neomycin
Citation Format: Kate R. Secombe, Imogen A. Ball, Anthony D. Wignall, Emma Bateman, Irmina Diala, Alshad S. Lalani, Joanne M. Bowen. Antibiotic treatment targeting gram negative bacteria prevents neratinib-induced diarrhea in rats abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1453.
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