Puma Biotechnology (United States)

Abstract Introduction: Small cell lung cancer (SCLC) accounts for 10-15% of lung cancers and is the most aggressive subtype. Approximately 16% of SCLCs contain mutations in the KRAS oncogene. The KRASG12C activating mutation is now clinically targetable. There are limited SCLC models containing the KRASG12C mutation available to generate pre-clinical rationale for clinical studies. This study aimed to develop a protocol for the insertion of the KRASG12C mutation in the KRAS wild-type SCLC cell line NCI-H1048 using CRISPR/Cas9 technology. Methods: A protospacer adjacent motif (PAM) was identified near the KRASG12C mutation site and a single guide RNA (sgRNA) was designed using SnapGene software. The efficiency and specificity of the sgRNA were determined using CRISPRon and CRISPRoff webservers (https://rth.dk/resources/crispr/). A repair template containing the nucleotide substitution was created. The CRISPR/Cas9 plasmid pSpCas9(BB)-2A-GFP (PX458, Addgene) was assembled with a single-stranded DNA oligo matching the sgRNA sequence. The NCI-H1048 cell line was transfected with the DNA assembly product (pKRASm) or the PX548 empty plasmid (control) using Lipofectamine 3000. Cells were sorted by GFP fluorescence 3 days after transfection using a BD FACS Aria III cell sorter. pKRASm- and PX458-transfected cells were seeded for colony formation in 10 cm tissue culture plates. Several pKRASm and PX458 clones were isolated and Sanger sequenced to determine KRASG12C mutation status. Codons affected by genetic alterations were determined by DNA local alignment using SnapGene software. Cell line growth rates were assessed over 7 days using the Incucyte® live-imaging system. Results: The transfection efficiency in NCI-H1048 was 0.6% for pKRASm and 1.2% for PX458. No clones with a KRASG12C mutation were isolated. However, two clones had alterations in the KRAS exon 2 sequence. Clone NCI-H1048-29 contained a 15bp in-frame deletion (impacting codons 9-13), and clone NCI-H1048-32 had heterozygous alleles from position 27 (impacting codons 9-37). The regions affected are the P-loop (codons 10-14, phosphate-binding loop) and switch I (codons 30-40, binding interface for KRAS effectors and regulators). The growth of clones NCI-H1048-29 and NCI-H1048-32 was compared to a control clone (PX458-transfected NCI-H1048-9). The KRAS alteration significantly reduced the growth of NCI-H1048-29 (p=0.023) and NCI-H1048-32 (p=0.004) compared to the NCI-H1048-9 control confirming phenotypic impact of the KRAS alterations. Conclusion: The CRISPR/Cas9 method employed resulted in the creation of novel SCLC cell lines with KRAS alterations, but further optimization of the protocol is needed to introduce KRAS point mutations. The CRISPR/Cas9-edited cell lines created will be used for further in vitro study of KRAS biology in SCLC. Citation Format: Myra Elise Castel, Neil T. Conlon, Georg F. Bischof, Ke Wei Gong, Neil A. O'Brien, John P. Crown, Denis M. Collins. Using CRISPR/Cas9 to edit KRAS in a small cell lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6846.

BackgroundHER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. Here we report that neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, has strong anti-proliferative effects against models of HER2-positive breast cancer that are innately resistant to trastuzumab or have acquired resistance to neratinib. MethodsNeratinib plus dasatinib was examined in a panel of 20 breast cancer cell lines, including HER2-positive, estrogen-receptor-positive, triple negative, and acquired HER2-targeted therapy resistant models. Drug effects on migration and apoptosis induction was evaluated and signaling alterations were determined by reverse phase protein array (RPPA). In vivo efficacy was examined using orthotopically-implanted HCC1954 cells. ResultsSynergy was observed in cell lines innately resistant to trastuzumab, models with acquired resistance to neratinib, and in triple negative breast cancer cell lines. Further investigation showed that neratinib plus dasatinib induced apoptosis and inhibited cell migration to a greater degree than either drug alone. RPPA revealed that the combination caused suppression of key survival signaling through EGFR, Akt, and MAPK inhibition. In vivo, neratinib plus dasatinib was well tolerated and had a prolonged anti-tumor effect against HCC1954 xenografts. ConclusionsThis study provides a strong pre-clinical rationale for the clinical investigation neratinib and dasatinib in HER2+ breast cancer.

Abstract Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines. Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting. Results: N displayed nanomolar IC50 values (< 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410. Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed. Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1978.

Abstract COVID-19 vaccines, including mRNA-1273, have been rapidly developed and deployed. To develop a safe and effective vaccine, establishing the optimal dose is crucial, and is where modeling and simulation can be used to guide vaccine dose selection and development. We developed an immunostimulatory/immunodynamic (IS/ID) model to quantitatively characterize the neutralizing antibody titers elicited by mRNA-1273 obtained from three clinical studies and to predict the optimal vaccine dose for future pediatric trials. A 25-µg primary vaccine series was predicted to meet non-inferiority criteria in young children (aged 2-5 years) and infants (aged 6-23 months); geometric mean titers and geometric mean ratios for this dose level predicted using the IS/ID model a priori matched those observed in the pediatric clinical study. These findings demonstrate that IS/ID models represent a novel approach to guide data-driven clinical dose selection of vaccines.

AIMS 6 mercaptopurine (6MP) is the mainstay chemotherapy for acute lymphoblastic leukaemia (ALL) and is conventionally available as 50 mg tablets. This study aimed to evaluate the bioequivalence of a new 6MP Powder for Oral Suspension (PFOS) intended for paediatric use. Additionally, a virtual study with the obtained data was planned for determining a dose of the PFOS that matches tablet exposures and to confirm optimal drug levels in pediatrics. METHODS An open-label, randomized, two-treatment, two-period, two-sequence, single oral dose, crossover, bioequivalence study was conducted on 51 healthy subjects. A population pharmacokinetic (PopPK) model was developed using the data to perform simulations with various PFOS doses and select a bioequivalent dose. To simulate 6MP and 6 thioguanine (6TGN) exposures in pediatrics, a literature model for paediatric ALL patients, and allometrically scaled PK parameters were utilised. RESULTS The 6MP PFOS had 47% higher bioavailability compared to the reference product. Simulations using a two-compartmental PopPK model with dissolution and transit compartments showed that 40 mg of PFOS was found to be equivalent to the 50mg tablets. The simulated 6TGN concentrations in virtual paediatric patients were between 114 and 703.6 pmol/8x108 RBCs, which was within the range of values reported in paediatric ALL studies. CONCLUSION The study demonstrates that 40 mg dose of 6MP PFOS 10 mg/mL has the same extent of absorption as the 50 mg tablet which can be precisely administered in pediatrics. The study also demonstrates the role of modelling and simulation to perform virtual bioequivalence and paediatric studies.

"CLO23-051: Real-World Use of Neratinib in Patients With HER2-Positive Early-Stage Breast Cancer Following Prior Ado-Trastuzumab Emtansine (T-DM1) or Dual HER2 Blockade With Pertuzumab Plus Trastuzumab" published on 31 Mar 2023 by National Comprehensive Cancer Network.

<div>AbstractPurpose:<p>We wanted to determine the prognosis and the phenotypic characteristics of hormone receptor–positive advanced breast cancer tumors harboring an <i>ERBB2</i> mutation in the absence of a HER2 amplification.</p>Experimental Design:<p>We retrospectively collected information from the American Association of Cancer Research-Genomics Evidence Neoplasia Information Exchange registry database from patients with hormone receptor–positive, HER2-negative, <i>ERBB2</i>-mutated advanced breast cancer. Phenotypic and co-mutational features, as well as response to treatment and outcome were compared with matched control cases <i>ERBB2</i> wild type.</p>Results:<p>A total of 45 <i>ERBB2</i>-mutant cases were identified for 90 matched controls. The presence of an <i>ERBB2</i> mutation was not associated with worse outcome determined by overall survival (OS) from first metastatic relapse. No significant differences were observed in phenotypic characteristics apart from higher lobular infiltrating subtype in the <i>ERBB2</i>-mutated group. <i>ERBB2</i> mutation did not seem to have an impact in response to treatment or time-to-progression (TTP) to endocrine therapy compared with <i>ERBB2</i> wild type. In the co-mutational analyses, <i>CDH1</i> mutation was more frequent in the <i>ERBB2-</i>mutated group (FDR < 1). Although not significant, fewer co-occurring <i>ESR1</i> mutations and more <i>KRAS</i> mutations were identified in the <i>ERBB2</i>-mutated group.</p>Conclusions:<p><i>ERBB2</i>-activating mutation was not associated with a worse OS from time of first metastatic relapse, or differences in TTP on treatment as compared with a series of matched controls. Although not significant, differences in coexisting mutations (<i>CDH1</i>, <i>ESR1</i>, and <i>KRAS</i>) were noted between the <i>ERBB2</i>-mutated and the control group.</p></div>