FDA Approval Summary: Capmatinib and Tepotinib for the Treatment of Metastatic NSCLC Harboring MET Exon 14 Skipping Mutations or Alterations.

The FDA approved capmatinib and tepotinib on May 6, 2020, and February 3, 2021, respectively. Capmatinib is indicated for patients with metastatic non-small cell lung cancer (mNSCLC) whose tumors have a mutation leading to mesenchymal-epithelial transition (MET) exon 14 skipping as detected by an FDA-approved test. Tepotinib is indicated for mNSCLC harboring MET exon 14 skipping alterations. The approvals were based on trials GEOMETRY mono-1 (capmatinib) and VISION (tepotinib). In GEOMETRY mono-1, overall response rate (ORR) per Blinded Independent Review Committee (BIRC) was 68% [95% confidence interval (CI), 48-84] with median duration of response (DoR) 12.6 months (95% CI, 5.5-25.3) in 28 treatment-naïve patients and 41% (95% CI: 29, 53) with median DoR 9.7 months (95% CI, 5.5-13) in 69 previously treated patients with NSCLC with mutations leading to MET exon 14 skipping. In VISION, ORR per BIRC was 43% (95% CI: 32, 56) with median DoR 10.8 months (95% CI, 6.9-not estimable) in 69 treatment-naïve patients and 43% (95% CI, 33-55) with median DoR 11.1 months (95% CI, 9.5-18.5) in 83 previously-treated patients with NSCLC harboring MET exon 14 alterations. These are the first two therapies to be FDA approved specifically for patients with metastatic NSCLC with MET exon 14 skipping.

FDA Approval Summary: Selpercatinib for the Treatment of Lung and Thyroid Cancers with RET Gene Mutations or Fusions.

On May 8, 2020, the FDA granted accelerated approval to selpercatinib for (i) adult patients with metastatic RET fusion-positive non-small cell lung cancer (NSCLC), (ii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET-mutant medullary thyroid cancer who require systemic therapy, and (iii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET fusion-positive thyroid cancer who require systemic therapy and who are radioactive iodine refractory (if radioactive iodine is appropriate). Approval was granted on the basis of the clinically important effects on the overall response rate (ORR) with prolonged duration of responses observed in a multicenter, open-label, multicohort clinical trial (LIBRETTO-001, NCT03157128) in patients whose tumors had RET alterations. ORRs within the approved patient populations ranged from 64% [95% confidence interval (CI), 54-73] in prior platinum-treated RET fusion-positive NSCLC to 100% (95% CI, 63-100) in systemic therapy-naïve RET fusion-positive thyroid cancer, with the majority of responders across indications demonstrating responses of at least 6 months. The product label includes warnings and precautions for hepatotoxicity, hypertension, QT interval prolongation, hemorrhagic events, hypersensitivity, risk of impaired wound healing, and embryo-fetal toxicity. This is the first approval of a drug specifically for patients with RET alterations globally.

Gene expression signatures of breast cancer stem and progenitor cells do not exhibit features of Warburg metabolism.

INTRODUCTION: Cancers are believed to adapt to continual changes in glucose and oxygen availability by relying almost exclusively on glycolytic metabolism for energy (i.e. the Warburg effect). The process by which breast cancers sustain growth in avascular tissue is thought to be mediated via aberrant hypoxia response with ensuing shifts in glycolytic metabolism. Given their role in initiating and perpetuating tumors, we sought to determine whether breast cancer stem and progenitor cells play an instrumental role in this adaptive metabolic response. METHODS: Breast cancer stem/progenitor cells were isolated from invasive ductal carcinomas, and benign stem cells (SC) were isolated from reduction mammoplasty tissues. Relative expression of 33 genes involved in hypoxia and glucose metabolism was evaluated in flow cytometrically isolated stem and progenitor cell populations. Significance between cohorts and cell populations was determined using Student's 2-tailed t test. RESULTS: While benign stem/progenitor cells exhibited few significant inter-group differences in expression of genes involved in hypoxia regulation or glucose metabolism, breast cancer stem/progenitor cells demonstrated significant inter-group variability. Breast cancer stem/progenitor cells adapted to microenvironments through changes in stem cell numbers and transcription of glycolytic genes. One of four breast cancer stem/progenitor cells subpopulations exhibited an aerobic glycolysis gene expression signature. This subpopulation comprises the majority of the tumor and therefore best reflects invasive ductal carcinoma tumor biology. Although PI3K/AKT mutations are associated with increased proliferation of breast cancer cells, mutations in breast cancer stem/progenitor cells subpopulations did not correlate with changes in metabolic gene expression. CONCLUSIONS: The adaptive capacity of breast cancer stem/progenitor cells may enable tumors to survive variable conditions encountered during progressive stages of cancer growth.

Estrogen receptor, progesterone receptor, interleukin-6 and interleukin-8 are variable in breast cancer and benign stem/progenitor cell populations.

BACKGROUND: Estrogen receptor positive breast cancers have high recurrence rates despite tamoxifen therapy. Breast cancer stem/progenitor cells (BCSCs) initiate tumors, but expression of estrogen (ER) or progesterone receptors (PR) and response to tamoxifen is unknown. Interleukin-6 (IL-6) and interleukin-8 (IL-8) may influence tumor response to therapy but expression in BCSCs is also unknown. METHODS: BCSCs were isolated from breast cancer and benign surgical specimens based on CD49f/CD24 markers. CD44 was measured. Gene and protein expression of ER alpha, ER beta, PR, IL-6 and IL-8 were measured by proximity ligation assay and qRT-PCR. RESULTS: Gene expression was highly variable between patients. On average, BCSCs expressed 10-106 fold less ERα mRNA and 10-103 fold more ERß than tumors or benign stem/progenitor cells (SC). BCSC lin-CD49f-CD24-cells were the exception and expressed higher ERα mRNA. PR mRNA in BCSCs averaged 10-104 fold less than in tumors or benign tissue, but was similar to benign SCs. ERα and PR protein detection in BCSCs was lower than ER positive and similar to ER negative tumors. IL-8 mRNA was 10-104 higher than tumor and 102 fold higher than benign tissue. IL-6 mRNA levels were equivalent to benign and only higher than tumor in lin-CD49f-CD24-cells. IL-6 and IL-8 proteins showed overlapping levels of expressions among various tissues and cell populations. CONCLUSIONS: BCSCs and SCs demonstrate patient-specific variability of gene/protein expression. BCSC gene/protein expression may vary from that of other tumor cells, suggesting a mechanism by which hormone refractory disease may occur.

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

Cell-cell transmission of VSV-G pseudotyped lentivector particles.

Many replicating viruses, including HIV-1 and HTLV-1, are efficiently transmitted from the cell surface of actively infected cells upon contact with bystander cells. In a previous study, we reported the prolonged cell surface retention of VSV-G replication-deficient pseudotyped lentivector prior to endocytic entry. However, the competing kinetics of cell surface versus dissociation, neutralization or direct transfer to other cells have received comparatively little attention. Here we demonstrate that the relative efficiency of cell-cell surface transmission can outpace "cell-free" transduction at limiting vector input. This coincides with the prolonged half-life of cell bound vector but occurs, unlike HTLV-1, without evidence for particle aggregation. These studies suggest that cell-surface attachment stabilizes particles and alters neutralization kinetics. Our experiments provide novel insight into the underexplored cell-cell transmission of pseudotyped particles.

RNA trafficking by acute myelogenous leukemia exosomes.

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML.

Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system.

Cell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

Entry kinetics and cell-cell transmission of surface-bound retroviral vector particles.

BACKGROUND: Transduction with recombinant HIV-1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. METHODS: To examine the underlying mechanism in more detail, we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. RESULTS: The investigation of synchronized transduction reveals cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1 degrees cell correlate inversely with the magnitude of transfer to 2 degrees targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell-type specific abundance of extracellular matrix fibronectin. Residual particle-extracellular fibronectin matrix binding and 2 degrees transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. CONCLUSIONS: Although cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. The present study highlights the inadvertent consequences for cell entry and cell-cell transfer.

Cellular microvesicle pathways can be targeted to transfer genetic information between non-immune cells.

Eukaryotic cell communication is based on protein signaling cascades that require direct cell-cell apposition, or receptor engagement by secreted molecules. The transmission of genetic information is thought to be uncommon, apart from recent reports of exosomal RNA transfer in immune and glioblastoma cells. We wished to examine if existing microvesicle pathways could be directly targeted for the horizontal transfer of RNA genomes in less specialized cell types. Using replication-deficient retrovirus vector, studies herein confirm that a range of cells routinely sequester a small population of these RNA genomes in a non-canonical compartment, refractory to antibody neutralization and unaffected by specific pharmacological inhibition of pathways involved in conventional viral trafficking. Our experiments further reveal the cytoplasmic colocalization of vector genomes with tetraspanin proteins as well as the PI-3-kinase sensitive trafficking and subsequent transmission to 2 degrees targets. Collectively, our results indicate a scalable process whereby cells route vector genomes to multivesicular bodies (MVB) for cytoplasmic trafficking and exosomal release. Our findings imply that cells can serve to deliver recombinant payload, targeted for the stable genetic modification of 2 degrees target cells.

CXCR4 induction in hematopoietic progenitor cells from Fanca(-/-), -c(-/-), and -d2(-/-) mice.

OBJECTIVE: Bone marrow failure is a near-universal occurrence in patients with Fanconi anemia (FA) and is thought to result from exhaustion of the hematopoietic stem cell (HSC) pool. Retrovirus-mediated expression of the deficient protein corrects this phenotype and makes FA a candidate disease for HSC-directed gene therapy. However, inherent repopulation deficits and stem cell attrition during conventional transduction culture prevent therapeutic chimerism. MATERIALS AND METHODS: We previously reported rapid transduction protocols to limit stem cell losses after ex vivo culture. Here we describe a complementary strategy intended to improve repopulation through upregulation of chemokine receptor (CXCR) 4, a principal factor in hematopoietic homing. RESULTS: Using murine models with transgenic disruption of Fanca, -c, and -d2, we found that c-kit(+) and sca-1(+) progenitor cells express levels of CXCR4 comparable with those of wild-type littermates. Lineage-depleted progenitor populations rapidly upregulated CXCR4 transcript and protein in response to cytokine stimulation or hypoxia, regardless of genotype. Hypoxia conditioning of lineage-depleted Fancc(-/-) progenitors also reduced oxidative stress, improved in vitro migration and led to improved chimerism in myeloablated recipients after transplantation. CONCLUSION: These studies provide evidence that CXCR4 regulation in progenitor cells from transgenic mice representing multiple FA genotypes is intact and that modulation of homing offers a potential strategy to offset the FA HSC repopulation deficiency.

High frequency induction of CC to TT tandem mutations in DNA repair-proficient mammalian cells.

The CC to TT tandem mutation is induced by UV radiation exposure, though at relatively low frequencies when compared with the more commonly induced C to T mutation. Induction of the tandem mutation by UV is enhanced in mammalian cells with certain genetic deficiencies; however, conditions have not been described in which the frequency of this mutation is enhanced in DNA repair-proficient mammalian cells. For this study, an integrated construct that detects C to T and CC to TT mutations at a single codon in mouse Aprt was used to examine UVB mutagenesis under various conditions. Oxidative stress, in the form of intracellular hydrogen peroxide, increased the frequency of UVB-induced CC to TT mutations. Surprisingly, exposure of the cells to two antioxidants (N-acetylcysteine and trolox), either alone or in combination, also enhanced UVB induction of CC to TT tandem mutations. These results demonstrate, for the first time, that the frequency of UVB-induced CC to TT tandem mutations can be enhanced dramatically in DNA repair-proficient mammalian cells, and suggest that the enhancing effect does not require direct damage to DNA.

The frequency of CC to TT tandem mutations in mismatch repair-deficient cells is increased in a cytosine run.

Mononucleotide runs are hot spots for frameshift mutations in mismatch repair (MMR)-deficient cells. However, a role for mononucleotide runs in the formation of base pair substitutions has not been tested. Previously, we demonstrated that ultraviolet radiation C (UVC)- or reactive oxygen species-induced CC to TT tandem mutations are markedly enhanced in MMR-deficient cells. The target for the mutational analysis was two cytosines in a run of five cytosines (5C) within mouse Aprt. Because mutation from C to T for either or both of the two critical cytosines created a codon yielding a functional Aprt protein, this assay allowed both single and tandem substitutions to be quantified and the relative ratios compared. To determine if the cytosine run increased the frequency of single and/or tandem base pair substitutions, alternative constructs were created in which the cytosine run was disrupted by flanking the target cytosines with either thymines (2Cpyr) or adenines (2Cpur). Disruption of the cytosine run dramatically decreased the frequency of UVC-induced tandem mutations in the 2Cpyr and 2Cpur constructs, as compared with the 5C construct. Moreover, CC to TT tandem mutations occurred spontaneously or were induced by oxidative stress only within the 5C construct. These results demonstrate that CC to TT tandem mutations in MMR-deficient cells form more readily in a homocytosine run than in a sequence limited to two cytosines.

Oxidative mutagenesis, mismatch repair, and aging.

A PubMed search for the term "oxidative stress" yields over 29,000 articles published on the subject over the past 10 years; more than 2000 of these articles also include the term "aging" in their title or abstract. Many theories of aging predict causal roles for oxidative stress in the myriad of pathological changes that occur as a function of age, including an increasing propensity to develop cancer. A possible link between aging and cancer is the induction and accumulation of somatic mutations caused by oxidative stress. This Review focuses on small mutational events that are induced by oxidative stress and the role of mismatch repair (MMR) in preventing their formation. It also discusses a possible inhibitory effect of oxidative stress on MMR. We speculate that a synergistic interaction between oxidative damage to DNA and reduced MMR levels will, in part, account for an accumulation of small mutational events, and hence cancer, with aging.

A role for Pms2 in the prevention of tandem CC --> TT substitutions induced by ultraviolet radiation and oxidative stress.

DNA mismatch repair (MMR) is important for preventing base-pair substitutions caused by spontaneous or damage-related DNA polymerase errors. We have used a reversion assay based on mouse Aprt to investigate the role of MMR in preventing ultraviolet radiation (UV) and oxidative stress induced tandem CC --> TT base pair substitutions in cultured mammalian cells. The reversion construct used for this assay can detect both C --> T and CC --> TT mutational events. Most spontaneous mutations in Pms2-deficient cells were single C --> T substitutions (88%), with the remainder being tandem CC --> TT substitutions (12%). The percentage of tandem CC --> TT substitutions rose to 64% and 94% for Pms2-deficient cells exposed to UV and a mixture of hydrogen peroxide and metals (Cu/Fe), respectively. Exposure to hydrogen peroxide alone or metals alone did not induce the tandem substitutions, nor did treatment of the cells with the alkylating agent ethylmethane sulfonate, which induces G --> A substitutions on the opposite strand. Tandem CC --> TT substitutions were also induced by UV irradiation and the hydrogen peroxide/metal mixture in Pms2-proficient cells, but at frequencies significantly lower than those observed in the Pms2-deficient cells. We conclude that mismatch repair plays an important role in preventing tandem CC --> TT substitutions induced by certain genotoxin exposures.