FDA Approval Summary: Pertuzumab, Trastuzumab, and Hyaluronidase-zzxf Injection for Subcutaneous Use in Patients with HER2-positive Breast Cancer.

On June 29, 2020, the FDA approved pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection (Phesgo) for the treatment of patients with HER2-positive early-stage and metastatic breast cancer. Patients should be selected for therapy based on an FDA-approved companion diagnostic test. Approval was primarily based on the FeDeriCa trial, a randomized, open-label, multicenter comparability study of pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection compared with intravenous pertuzumab and intravenous trastuzumab administered in the neoadjuvant and adjuvant settings with chemotherapy for the treatment of patients with early breast cancer. The pharmacokinetic endpoints were, first, to demonstrate that the exposure of subcutaneous pertuzumab was not inferior to that of intravenous pertuzumab, and then to demonstrate that the exposure of subcutaneous trastuzumab was not inferior to that of intravenous trastuzumab. The primary endpoints were met with the observed lower limit of the two-sided 90% confidence intervals above the prespecified noninferiority margins. The most common adverse reactions were alopecia, nausea, diarrhea, anemia, and asthenia. The totality of the evidence demonstrated comparability of the subcutaneous product to intravenous, allowing for extrapolation and approval of all breast cancer indications for which intravenous trastuzumab and pertuzumab are approved.

Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-ß1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

Control of PD-L1 Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer.

Alterations in EGFR, KRAS, and ALK are oncogenic drivers in lung cancer, but how oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. Immunosuppression likely contributes to lung cancer, because drugs that inhibit immune checkpoints like PD-1 and PD-L1 have clinical benefit. Here, we show that activation of the AKT-mTOR pathway tightly regulates PD-L1 expression in vitro and in vivo. Both oncogenic and IFNγ-mediated induction of PD-L1 was dependent on mTOR. In human lung adenocarcinomas and squamous cell carcinomas, membranous expression of PD-L1 was significantly associated with mTOR activation. These data suggest that oncogenic activation of the AKT-mTOR pathway promotes immune escape by driving expression of PD-L1, which was confirmed in syngeneic and genetically engineered mouse models of lung cancer where an mTOR inhibitor combined with a PD-1 antibody decreased tumor growth, increased tumor-infiltrating T cells, and decreased regulatory T cells.

Rapamycin prevents the development and progression of mutant epidermal growth factor receptor lung tumors with the acquired resistance mutation T790M.

Lung cancer in never-smokers is an important disease often characterized by mutations in epidermal growth factor receptor (EGFR), yet risk reduction measures and effective chemopreventive strategies have not been established. We identify mammalian target of rapamycin (mTOR) as potentially valuable target for EGFR mutant lung cancer. mTOR is activated in human lung cancers with EGFR mutations, and this increases with acquisition of T790M mutation. In a mouse model of EGFR mutant lung cancer, mTOR activation is an early event. As a single agent, the mTOR inhibitor rapamycin prevents tumor development, prolongs overall survival, and improves outcomes after treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI). These studies support clinical testing of mTOR inhibitors in order to prevent the development and progression of EGFR mutant lung cancers.

SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines.

OBJECTIVES: Primary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors. MATERIALS AND METHODS: Immunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed. RESULTS AND CONCLUSION: Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFR(L858R/T790M) mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.

GSK-3α promotes oncogenic KRAS function in pancreatic cancer via TAK1-TAB stabilization and regulation of noncanonical NF-κB.

Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently, TGF-ß-activated kinase 1 (TAK1), an upstream regulator of IκB kinase (IKK), which controls canonical NF-κB signaling, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS-mutant colorectal cancer cell growth and survival. Here, we show that mutant KRAS upregulates glycogen synthase kinase 3α (GSK-3α), leading to its interaction with TAK1 to stabilize the TAK1-TAB complex to promote IKK activity. In addition, GSK-3α is required for promoting critical noncanonical NF-κB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3α as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-κB signaling pathways.

Maintenance of constitutive IkappaB kinase activity by glycogen synthase kinase-3alpha/beta in pancreatic cancer.

Constitutive nuclear factor kappaB (NF-kappaB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3beta (GSK-3beta) plays a key role in maintaining basal NF-kappaB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3beta facilitates constitutive NF-kappaB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3alpha and GSK-3beta) in regulating NF-kappaB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-kappaB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IkappaB kinase (IKK) subunits are not equally required to regulate pancreatic cancer-associated NF-kappaB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-kappaB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-kappaB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.

Complex cardiac Nkx2-5 gene expression activated by noggin-sensitive enhancers followed by chamber-specific modules.

We previously reported that an Nkx2-5-GFP bacterial artificial chromosome in transgenic mice recapitulated the endogenous gene activity in the heart. Here, we identified three additional previously uncharacterized distal enhancer modules of Nkx2-5: UH6, which directed transgene expression in the right ventricle, interventricular septum, and atrial ventricular canal; UH5, which directed expression in both atria; and UH4, which directed transgene expression in tongue muscle. Nkx2-5 enhancers drive cardiogenic gene activity from the earliest progenitors to the late-stage embryonic heart, reside within its 27 kb of 5' flanking sequences, organized in a tandem array. Nkx2-5 enhancers involved with stomach-, tongue-, and chamber-restricted expression displayed lacZ transgene activity and chromatin histone acetylation patterns consistent with tissue-specific expression. An examination of Nkx2-5 gene activity in murine embryonic stem cells converted to beating embryoid bodies showed that only the proximal active region 2 and GATA-Smad enhancers were chromatin-remodeled. Chromatin remodeling of active region 2 and GATA-Smad enhancers were blunted by noggin coexpression, which indicated dependence on bone morphogenetic protein signaling for their chromatin activation during activation of Nkx2-5 expression.

Glycogen synthase kinase 3beta functions to specify gene-specific, NF-kappaB-dependent transcription.

Loss of glycogen synthase kinase 3beta (GSK-3beta) in mice results in embryonic lethality via hepatocyte apoptosis. Consistent with this result, cells from these mice have diminished nuclear factor kappaB (NF-kappaB) activity, implying a functional role for GSK-3beta in regulating NF-kappaB. Here, we have explored mechanisms by which GSK-3beta may control NF-kappaB function. We show that cytokine-induced IkappaB kinase activity and subsequent phosphorylation of IkappaBalpha, p105, and p65 are not affected by the absence of GSK-3beta activity. Furthermore, nuclear accumulation of p65 following tumor necrosis factor treatment is unaffected by the loss of GSK-3beta. However, NF-kappaB DNA binding activity is reduced in GSK-3beta null cells and in cells treated with a pharmacological inhibitor of GSK-3. Expression of certain NF-kappaB-regulated genes, such as IkappaBalpha and macrophage inflammatory protein 2, is minimally affected by the absence of GSK-3beta. Conversely, we have identified a subset of NF-kappaB-regulated genes, including those for interleukin-6 and monocyte chemoattractant protein 1, that require GSK-3beta for efficient expression. We show that efficient localization of p65 to the promoter regions of the interleukin-6 and monocyte chemoattractant protein 1 genes following tumor necrosis factor alpha treatment requires GSK-3beta. Therefore, GSK-3beta has profound effects on transcription in a gene-specific manner through a mechanism involving control of promoter-specific recruitment of NF-kappaB.

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes.

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)-BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15.

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.

Hemodynamic parameters in blood vessels in choroidal melanoma xenografts and rat choroid.

PURPOSE: Choroidal melanoma is the most common primary ocular cancer among the adult population. To avoid enucleation, there has been a concerted effort to develop therapies that spare the affected eye and the patient's vision. Blood flow helps shape the tumor's microenvironment, plays a key role in the tumor's response to many different types of therapy, and is necessary for delivery of chemotherapeutic agents. To rationally design new therapies and optimize existing treatments, it is essential to learn as much as possible about blood flow and the microcirculation in this tumor. In recent years, much has been discovered about the anatomy of the microvasculature and the dynamics of overall blood flow in choroidal melanoma, but little is known about the factors that determine microvascular blood flow. In this study hemodynamic parameters in individual microvessels of a human choroidal melanoma xenograft were compared with those same parameters in a normal rat choroid. METHODS: Nude, athymic WAG/RijHs-rnu rats were used in this study. The human choroidal melanoma cell line OCM-1 was used to grow solid tumors subcutaneously in the flanks of donor rats. Small pieces of these tumors were then implanted into the choroidal space of recipient rats. After 6 to 8 weeks, the rats were anesthetized with a subcutaneous injection of urethane, and the sclera was exposed. Rhodamine-labeled liposomes and red blood cells (RBCs) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were injected intravenously. Epifluorescent, intravital microscopy was used to visualize the flow of fluorescent RBCs through individual vessels in the choroid or tumor. Flow through multiple vessels was recorded on videotape for later analysis. From the recordings, RBC flux, RBC velocity (V(c)), and microvascular hematocrit (HCT(m)) were determined. Similar experiments were performed in rats with no tumor growth, and these same parameters were calculated in normal choroidal vessels. RBC flow was characterized in 55 vessels in six OCM-1 tumors and in 153 choroidal vessels in five non-tumor-bearing rats. RESULTS: RBC flux was higher in larger tumor vessels (>30 micro m in diameter) compared with similarly sized choroidal vessels. There were no differences in the velocities of RBCs through the two types of vessels. HCT(m) was significantly higher in medium-sized (>20 micro m in diameter) and larger tumor vessels compared with normal choroidal vessels. CONCLUSIONS: These experiments demonstrate differences between hemodynamic parameters in normal choroidal microvessels and microvessels in choroidal melanoma in this animal model. Because HCT(m) is a key determinant of apparent viscosity, abnormally high HCT(m) in the tumor vessels would increase vascular resistance and decrease flow. This could have a negative impact on the tumor oxygen levels and on the ability to deliver drugs effectively. On the contrary, higher local HCT(m) has also been shown to increase oxygen delivery. The impact and interplay of these two effects on tumor oxygenation remain to be determined.