TKI Type Switching Overcomes ROS1 L2086F in ROS1 Fusion-Positive Cancers.

Purpose: Despite the robust efficacy of ROS1 tyrosine kinase inhibitors (TKIs) in ROS1-positive non-small cell lung cancer, TKI resistance continues to hamper durability of the therapeutic response. The resistance liabilities of next-generation ROS1 TKI are sparsely characterized. Design: We compared the activity of type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) to the type II TKIs (cabozantinib and merestinib), and to the type I FLT3 inhibitor, gilteritinib, in CD74-ROS1 wildtype and F2004C, L2026M, G2032R, or L2086 mutant Ba/F3 cells. The findings from the Ba/F3 cell model were confirmed using NIH3T3 colony formation assays and in vivo tumor growth. CRISPR/Cas9 gene editing was used to generate isogenic wildtype and L2086F mutant TPM3-ROS1 expressing patient-derived cell lines. These lines were used to further evaluate TKI activity using cell viability and immunoblotting methods. Molecular modeling studies enabled the characterization of structural determinants of TKI sensitivity in wildtype and mutant ROS1 kinase domains. We also report clinical cases of ROS1 TKI resistance that were treated with cabozantinib. Results: ROS1 L2086F mutant kinase is resistant to type I TKI including crizotinib, entrectinib, lorlatinib, repotrectinib, taletrectinib, while the type II TKI cabozantinib and merestinib retain activity. Additionally, we found that gilteritinib, a type I FLT3 inhibitor, inhibited wildtype and L2086F mutant ROS1, however ROS1 G2032R solvent front mutation imposed resistance. The specific binding poses adopted by cabozantinib in the DFG-out kinase conformation and gilteritinib in the DFG-in kinase, provide rationale for their activity in the ROS1 mutants. Clinical cases demonstrated response to cabozantinib in tumors developing TKI resistance due to the ROS1 L2086F mutation. Conclusion: Cabozantinib and gilteritinib effectively inhibit ROS1 L2086F. Clinical activity of cabozantinib is confirmed in patients with TKI-resistant, ROS1 L2086F mutant NSCLC. Gilteritinib may offer an alternative with distinct off-target toxicities, however further studies are required. Since cabozantinib and gilteritinib are multi-kinase inhibitors, there is a persistent unmet need for more selective and better-tolerated TKI to overcome ROS1 L2086F kinase-intrinsic resistance. Translational relevance: ROS1 L2086F is an emerging recurrent resistance mutation to type I ROS1 TKIs, including later generation TKIs. Here, we show corroborating preclinical and clinical evidence for the activity of the quinolone-based type II TKI, cabozantinib, in ROS1 L2086F resistance setting. In addition, we show activity of the pyrazine carboxamide-based type I TKI, gilteritinib, in ROS1 L2086F resistance, suggesting that gilteritinib could be another option for ROS1 L2086F TKI-resistant patients. This study represents the first comprehensive report of ROS1 L2086F in the context of later-generation TKIs, including the macrocyclic inhibitors.

Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors.

ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI-sensitive oncogenic variants in cell-based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA-approved ROS1-TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA-approved ROS1-TKIs.

Functional impact and targetability of PI3KCA, GNAS, and PTEN mutations in a spindle cell rhabdomyosarcoma with MYOD1 L122R mutation.

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma, commonly harboring a gain-of-function L122R mutation in the muscle-specific master transcription factor MYOD1. MYOD1-mutated ssRMS is almost invariably fatal, and development of novel therapeutic approaches based on the biology of the disease is urgently needed. MYOD1 L122R affects the DNA-binding domain and is believed to confer MYC-like properties to MYOD1, driving oncogenesis. Moreover, the majority of the MYOD1-mutated ssRMS harbor additional alterations activating the PI3K/AKT pathway. It is postulated that the PI3K/AKT pathway cooperates with MYOD1 L122R. To address this biological entity, we established and characterized a new patient-derived ssRMS cell line OHSU-SARC001, harboring MYOD1 L122R as well as alterations in PTEN, PIK3CA, and GNAS We explored the functional impact of these aberrations on oncogenic signaling with gain-of-function experiments in C2C12 murine muscle lineage cells. These data reveal that PIK3CAI459_T462del, the novel PIK3CA variant discovered in this patient specimen, is a constitutively active kinase, albeit to a lesser extent than PI3KCAE545K, a hotspot oncogenic mutation. Furthermore, we examined the effectiveness of molecularly targeted PI3K/AKT/mTOR and RAS/MAPK inhibitors to block oncogenic signaling and suppress the growth of OHSU-SARC001 cells. Dual PI3K/mTOR (LY3023414, bimiralisib) and AKT inhibitors (ipatasertib, afuresertib) induced dose-dependent reductions in cell growth. However, mTOR-selective inhibitors (everolimus, rapamycin) alone did not exert cytotoxic effects. The MEK1/2 inhibitor trametinib did not impact proliferation even at the highest doses tested. Our data suggest that molecularly targeted strategies may be effective in PI3K/AKT/mTOR-activated ssRMS. Taken together, these data highlight the importance of utilizing patient-derived models to assess molecularly targetable treatments and their potential as future treatment options.

Discovery and Characterization of Recurrent, Targetable ALK Fusions in Leiomyosarcoma.

Soft-tissue sarcomas such as leiomyosarcoma pose a clinical challenge because systemic treatment options show only modest therapeutic benefit. Discovery and validation of targetable vulnerabilities is essential. To discover putative kinase fusions, we analyzed existing transcriptomic data from leiomyosarcoma clinical samples. Potentially oncogenic ALK rearrangements were confirmed by application of multiple RNA-sequencing fusion detection algorithms and FISH. We functionally validated the oncogenic potential and targetability of discovered kinase fusions through biochemical, cell-based (Ba/F3, NIH3T3, and murine smooth muscle cell) and in vivo tumor modeling approaches. We identified ALK rearrangements in 9 of 377 (2.4%) patients with leiomyosarcoma, including a novel KANK2-ALK fusion and a recurrent ACTG2-ALK fusion. Functional characterization of the novel ALK fusion, KANK2-ALK, demonstrates it is a dominant oncogene in Ba/F3 or NIH3T3 model systems, and has tumorigenic potential when introduced into smooth muscle cells. Oral monotherapy with targeted ALK kinase inhibitor lorlatinib significantly inhibits tumor growth and prolongs survival in a murine model of KANK2-ALK leiomyosarcoma. These results provide the first functional validation of a targetable oncogenic kinase fusion as a driver in a subset of leiomyosarcomas. Overall, these findings suggest that some soft-tissue sarcomas may harbor previously unknown kinase gene translocations, and their discovery may propel new therapeutic strategies in this treatment-refractory cancer. IMPLICATIONS: A subset of leiomyosarcomas harbor previously unrecognized oncogenic ALK fusions that are highly responsive to ALK inhibitors and thus these data emphasize the importance of detailed genomic investigations of leiomyosarcoma tumors.

Gonadotroph-specific expression of the human follicle stimulating hormone beta gene in transgenic mice.

A paucity of in vitro models has hampered studies of molecular mechanisms of FSH subunit gene expression. Consequently, we used an in vivo transgenic strategy to map the location of regulatory elements in the cloned 10 kb human FSHbeta gene. Analyses of transgenic mouse lines revealed that successive 5' truncations of the hFSHbeta promoter region to -350 bp relative to the transcriptional initiation site retained gonadotroph-specific expression and the sexually dimorphic pattern of male greater than female FSHbeta mRNA levels found normally in rodent pituitary. Truncation of the 3' flanking sequences from positions +3142 to +2138 bp relative to the translational stop codon in exon 3 resulted in a complete loss of transgene expression, suggesting the presence of critical regulatory elements mapping to the 1 kb genomic segment downstream of position +2138, in addition to the proximal 5' promoter elements. In silico phylogenetic comparisons of mammalian FSHbeta genes revealed five islands of highly conserved sequence homology corresponding precisely to the proximal 5' promoter region, exon 2, the 5' translated region of exon 3, and two regions at the 3' untranslated end of exon 3 that include putative polyadenylation and transcriptional termination signals. Sequence analyses of the 5' proximal promoter revealed the presence of several putative homeodomain binding sites as well as GATA, SMAD, AP-1, NF-1, NF-Y and steroid hormone transcription factor binding sites within the highly conserved -350 bp promoter region. Notably absent from these 5' sequences, however, are consensus binding sites for either Egr-1 or Lim-2 transcription factors known to be critical for the gonadotroph-specific expression of the LHbeta gene. These findings support the hypothesis that one of the mechanisms underlying the differential regulation of the LHbeta, FSHbeta, and common alpha-gonadotropin subunits within pituitary gonadotrophs may be differences in sequence-specific binding requirements for distinct combinations of transcription factors.

Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection.

The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.

Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.