Translational insights and overall survival in the U31402-A-U102 study of patritumab deruxtecan (HER3-DXd) in EGFR-mutated NSCLC.

BACKGROUND: Human epidermal growth factor receptor 3 (HER3) is broadly expressed in non-small-cell lung cancer (NSCLC) and is the target of patritumab deruxtecan (HER3-DXd), an antibody-drug conjugate consisting of a HER3 antibody attached to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. U31402-A-U102 is an ongoing phase I study of HER3-DXd in patients with advanced NSCLC. Patients with epidermal growth factor receptor (EGFR)-mutated NSCLC that progressed after EGFR tyrosine kinase inhibitor (TKI) and platinum-based chemotherapy (PBC) who received HER3-DXd 5.6 mg/kg intravenously once every 3 weeks had a confirmed objective response rate (cORR) of 39%. We present median overall survival (OS) with extended follow-up in a larger population of patients with EGFR-mutated NSCLC and an exploratory analysis in those with acquired genomic alterations potentially associated with resistance to HER3-DXd. PATIENTS AND METHODS: Safety was assessed in patients with EGFR-mutated NSCLC previously treated with EGFR TKI who received HER3-DXd 5.6 mg/kg; efficacy was assessed in those who also had prior PBC. RESULTS: In the safety population (N = 102), median treatment duration was 5.5 (range 0.7-27.5) months. Grade ≥3 adverse events occurred in 76.5% of patients; the overall safety profile was consistent with previous reports. In 78/102 patients who had prior third-generation EGFR TKI and PBC, cORR by blinded independent central review (as per RECIST v1.1) was 41.0% [95% confidence interval (CI) 30.0% to 52.7%], median progression-free survival was 6.4 (95% CI 4.4-10.8) months, and median OS was 16.2 (95% CI 11.2-21.9) months. Patients had diverse mechanisms of EGFR TKI resistance at baseline. At tumor progression, acquired mutations in ERBB3 and TOP1 that might confer resistance to HER3-DXd were identified. CONCLUSIONS: In patients with EGFR-mutated NSCLC after EGFR TKI and PBC, HER3-DXd treatment was associated with a clinically meaningful OS. The tumor biomarker characterization comprised the first description of potential mechanisms of resistance to HER3-DXd therapy.

Fatal myeloproliferation, induced in mice by TEL/PDGFbetaR expression, depends on PDGFbetaR tyrosines 579/581.

The t(5;12)(q33;p13) translocation associated with chronic myelomonocytic leukemia (CMML) generates a TEL/PDGFbetaR fusion gene. Here, we used a murine bone marrow transplant (BMT) assay to test the transforming properties of TEL/PDGFbetaR in vivo. TEL/PDGFbetaR, introduced into whole bone marrow by retroviral transduction, caused a rapidly fatal myeloproliferative disease that closely recapitulated human CMML. TEL/PDGFbetaR transplanted mice developed leukocytosis with Gr-1(+) granulocytes, splenomegaly, evidence of extramedullary hematopoiesis, and bone marrow fibrosis, but no lymphoproliferative disease. We assayed mutant forms of the TEL/PDGFbetaR fusion protein - including 8 tyrosine to phenylalanine substitutions at phosphorylated PDGFbetaR sites to which various SH2 domain-containing signaling intermediates bind - for ability to transform hematopoietic cells. All of the phenylalanine (F-) mutants tested conferred IL-3-independence to a cultured murine hematopoietic cell line, but, in the BMT assay, different F-mutants displayed distinct transforming properties. In transplanted animals, tyrosines 579/581 proved critical for the development of myeloproliferative phenotype. F-mutants with these residues mutated showed no sign of myeloproliferation but instead developed T-cell lymphomas. In summary, TEL/PDGFbetaR is necessary and sufficient to induce a myeloproliferative disease in a murine BMT model, and PDGFbetaR residues Y579/581 are required for this phenotype.

Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation.

TEL-JAK2 fusion proteins, which are a result of t(9;12)(p24;p13) translocations associated with human leukemia, activate Stat5 in vitro and in vivo and cause a myelo- and lymphoproliferative disease in a murine bone marrow transplant model. We report that Socs-1, a member of the SOCS family of endogenous inhibitors of JAKs and STATs, inhibits transformation of Ba/F3 cells by TEL-JAK2 but has no effect on Ba/F3 cells transformed by BCR-ABL, TEL-ABL, or TEL-platelet-derived growth factor receptor beta. TEL-JAK2, in addition to activating Stat5, associates with Shc and Grb2 and induces activation of Erk2, and expression of Socs-1 inhibits engagement of each of these signaling molecules. TEL-JAK2 kinase activity is inhibited by Socs-1, as assessed by in vitro kinase assays. In addition, Socs-1 induces proteasomal degradation of TEL-JAK2. Mutational analysis indicates that the SOCS box of Socs-1 is required for proteasomal degradation and for abrogation of growth of TEL-JAK2-transformed cells. Furthermore, murine bone marrow transplant assays demonstrate that expression of Socs-1 prolongs latency of TEL-JAK2-mediated disease in vivo. Collectively, these data indicate that Socs-1 inhibits TEL-JAK2 in vitro and in vivo through inhibition of kinase activity and induction of TEL-JAK2 protein degradation.

KIAA1509 is a novel PDGFRB fusion partner in imatinib-responsive myeloproliferative disease associated with a t(5;14)(q33;q32).

We report the cloning of a novel PDGFRB fusion gene partner in a patient with a chronic myeloproliferative disorder characterized by t(5;14)(q33;q32), who responded to treatment with imatinib mesylate. Fluorescence in situ hybridization demonstrated that PDGFRB was involved in the translocation. Long distance inversion PCR identified KIAA1509 as the PDGFRB fusion partner. KIAA1509 is an uncharacterized gene with a predicted coiled-coil oligomerization domain with homology to the HOOK family of proteins. The predicted KIAA1509-PDGFRbeta fusion protein contains the KIAA1509 coiled-coil domain fused to the cytoplasmic domain of PDGFRbeta that includes the tyrosine kinase domain. Imatinib therapy resulted in rapid normalization of the patient's blood counts, and subsequent bone marrow biopsies and karyotypic analysis were consistent with sustained complete remission.

Activation of a histone H1 kinase by tyrosine phosphorylation in v-src-transformed fibroblasts.

Using anti-phosphotyrosine immunoaffinity chromatography, we have searched for serine/threonine kinases that are directly regulated by tyrosine phosphorylation in v-src-transformed rat 3Y1 fibroblasts. Tyrosine phosphoprotein preparations from v-src-transformed cells contain a kinase activity that phosphorylates histone H1 in vitro on serine residues and this activity is present at a 20-fold greater level than that in parental cell preparations. This activity elutes from a MonoQ FPLC column as a single peak and gel filtration chromatography suggests that the kinase has a molecular mass of approximately 55 kDa. Tyrosine phosphatase treatment inactivates the histone H1 kinase and this result indicates that the specific activity of the kinase is regulated by tyrosine phosphorylation. Experiments with cells transformed with a temperature-sensitive mutant of the v-src oncogene demonstrate that the tyrosine phosphorylation of the histone H1 kinase is an early event in v-src transformation. The kinase is distinct from known cdc2 family members that contain the PSTAIR motif, because the kinase can be separated almost completely from these proteins by immunoprecipitation with an antibody against p34cdc2. The profile of antibody reactivity and sensitivity to modulators of protein kinases suggests that this activity is distinct from known second messenger-regulated kinases and from previously characterized MAP kinases.

Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells.

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.

The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation.

Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.

S-methyl glutathione synthesis is catalyzed by the cheR methyltransferase in Escherichia coli.

The cheR methyltransferase, known to be necessary for the methyl esterification of receptors involved in chemotaxis, is shown to be essential to the synthesis of S-methyl glutathione from glutathione and S-adenosylmethionine in intact Escherichia coli. S-Methyl glutathione is not, however, found to be essential for chemotaxis. It is suggested that the synthesis of S-methyl glutathione may be due to a "parasitic" reaction of glutathione with S-adenosylmethionine bound to the methyltransferase.

K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.

Treatment of patients with recurrent and primary refractory acute myelogenous leukemia using mitoxantrone and intermediate-dose cytarabine: a pharmacologically based regimen.

BACKGROUND: Although chemotherapy can achieve a high rate of disease remission induction in patients with newly diagnosed acute myelogenous leukemia (AML), patients with recurrent or refractory AML generally have a poorer rate of response. This study assessed the utility of mitoxantrone and intermediate-dose cytarabine (Ara-C) in the treatment of patients with recurrent or refractory AML. METHODS: Forty-seven patients with recurrent or refractory AML were treated with Ara-C, 0.5 gm/m2, intravenously (i.v.) every 12 hours x 12 doses on Days 1-6 and mitoxantrone, 5 mg/m2, i.v. on Days 1-5. RESULTS: Twenty-nine of the 47 patients (62%) achieved a complete response. The median duration of disease remission was 112 days (range, 29 days- 8 years). Of the 25 patients age > or = 60 years, 19 (76%) had a complete disease remission and the median duration of disease remission in this group was 114 days (range, 33-370 days), although all patients subsequently developed a disease recurrence. The chemotherapy generally was well tolerated, with a mean duration of neutropenia of 31 days and a mean duration of thrombocytopenia of 33 days. Three patients died of infectious complications between 23-26 days after the initiation of chemotherapy, 1 patient died of sudden cardiac arrest 13 days after the initiation of chemotherapy, and 1 patient developed cutaneous desquamation. Three patients developed acute cerebellar dysfunction. CONCLUSIONS: The use of mitoxantrone and Ara-C is effective in the treatment of patients with recurrent and refractory AML. The subgroup of patients age > or = 60 years also had a high rate of disease remission induction with this regimen, and the regimen generally was well tolerated.

The TEL/PDGFbetaR fusion in chronic myelomonocytic leukemia signals through STAT5-dependent and STAT5-independent pathways.

The TEL/PDGFbetaR gene, which encodes a fusion protein containing the ETS-family member TEL fused to the protein-tyrosine kinase domain of the platelet-derived growth factor receptor-beta (PDGFbetaR), confers interleukin 3 (IL-3)-independent growth on Ba/F3 hematopoietic cells. TEL/PDGFbetaR mutants have been generated that contain tyrosine-to-phenylalanine (Tyr-->Phe) substitutions at phosphorylation sites present in the native PDGFbetaR to assess the role of these sites in cell transformation by TEL/PDGFbetaR. Similar to previous findings in a murine bone marrow transplantation model, full transformation of Ba/F3 cells to IL-3-independent survival and proliferation required the TEL/PDGFbetaR juxtamembrane and carboxy terminal phosphorylation sites. In contrast to previous reports concerning comparable mutants in the native PDGFbetaR, each of the TEL/PDGFbetaR mutants is fully active as a protein-tyrosine kinase. Expression of the TEL/PDGFbetaR fusion protein causes hyperphosphorylation and activation of signal transducer and activator of transcription (STAT5), and this activation of STAT5 requires the juxtamembrane Tyr579 and Tyr581 in the TEL/PDGFbetaR fusion. Hyperphosphosphorylation of phospholipase Cgamma (PLCgamma) and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) requires the carboxy terminal tyrosine residues of TEL/PDGFbetaR. Thus, full transformation of Ba/F3 cells by TEL/PDGFbetaR requires engagement of PI3K and PLCgamma and activation of STAT5. Taken together with the growth properties of cells transformed by the TEL/PDGFbetaR variants, these findings indicate that a minimal combination of these signaling intermediates contributes to hematopoietic transformation by the wild-type TEL/PDGFbetaR fusion. (Blood. 2001;98:3390-3397)

H4(D10S170), a gene frequently rearranged in papillary thyroid carcinoma, is fused to the platelet-derived growth factor receptor beta gene in atypical chronic myeloid leukemia with t(5;10)(q33;q22).

The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase- polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor betaR (PDGFbetaR) fusion transcript that incorporated 5' sequences from H4 fused in frame to 3' PDGFbetaR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13(+) and CD14(+) cells but was not observed in CD3(+) or CD19(+) cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFbetaR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFbetaR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFbetaR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFbetaR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFbetaR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFbetaR, for efficient induction of factor-independent growth of Ba/F3 cells. (Blood. 2001;97:3910-3918)