The EMA Assessment of Asciminib for the Treatment of Adult Patients With Philadelphia Chromosome-Positive Chronic Myeloid Leukemia in Chronic Phase Who Were Previously Treated With at Least Two Tyrosine Kinase Inhibitors.

Asciminib is an allosteric high-affinity tyrosine kinase inhibitor (TKI) of the BCR-ABL1 protein kinase. This kinase is translated from the Philadelphia chromosome in chronic myeloid leukemia (CML). Marketing authorization for asciminib was granted on August 25, 2022 by the European Commission. The approved indication was for patients with Philadelphia chromosome-positive CML in the chronic phase which have previously been treated with at least 2 TKIs. Clinical efficacy and safety of asciminib were evaluated in the open-label, randomized, phase III ASCEMBL study. The primary endpoint of this trial was major molecular response (MMR) rate at 24 weeks. A significant difference in MRR rate was shown between the asciminib treated population and the bosutinib control group (25.5% vs. 13.2%, respectively, P = .029). In the asciminib cohort, adverse reactions of at least grade 3 with an incidence ≥ 5% were thrombocytopenia, neutropenia, increased pancreatic enzymes, hypertension, and anemia. The aim of this article is to summarize the scientific review of the application which led to the positive opinion by the European Medicines Agency's Committee for Medicinal Products for Human Use.

European Conditional Marketing Authorization in a Rapidly Evolving Treatment Landscape: A Comprehensive Study of Anticancer Medicinal Products in 2006-2020.

Since 2006, the European conditional marketing authorization (CMA) aims to facilitate timely patient access to medicinal products for which there is an unmet medical need by accepting less comprehensive data than normally required. The granting of CMA requires a positive benefit-risk balance, unmet medical needs to be fulfilled, likely submission of comprehensive data postauthorization, and the benefit of immediate availability to outweigh the risks of data noncomprehensiveness. Since its first use, more than half of all CMAs represent (hemato-)oncology indications. Therefore, we aimed to investigate the conditions in which CMA has been applied for anticancer medicinal products and whether they have changed over time. We retrospectively assessed the European public assessment reports of the 30 anticancer medicinal products granted CMA in 2006-2020 (51% of all 59 CMAs). Comparison of 2006-2013 to 2014-2020 highlighted increased proportions of proactively requested CMAs (+40%), medicinal products that addressed unmet medical needs by providing a major therapeutic advantage over authorized treatments (+38%), and orphan designated indications (+32%). In contrast, it showed decreased proportions of medicinal products for which a scientific advisory group was consulted (-55%) and phase III randomized controlled trial data were available (-38%). This suggests that applicants and the European Medicines Agency have learned how to use the CMA as a regulatory tool, among others, through better planning and proactive interaction. However, the increasing number of granted CMAs complicates the establishment of unmet medical need and the benefit-risk balance, especially in crowded indications and when only phase II uncontrolled trials are available.

A Comparison of Post-marketing Measures Imposed by Regulatory Agencies to Confirm the Tissue-Agnostic Approach.

There are currently four anti-cancer medicinal products approved for a tissue-agnostic indication. This is an indication based on a common biological characteristic rather than the tissue of origin. To date, the regulatory experience with tissue-agnostic approvals is limited. Therefore, we compared decision-making aspects of the first tissue-agnostic approvals between the Food and Drug Administration (FDA), European Medicines Agency (EMA) and Pharmaceuticals and Medical Devices Agency (PMDA). Post-marketing measures (PMMs) related to the tissue-agnostic indication were of specific interest. The main data source was the publicly available review documents. The following data were collected: submission date, approval date, clinical trials and datasets, and PMMs. At the time of data collection, the FDA and PMDA approved pembrolizumab, larotrectinib, and entrectinib for a tissue-agnostic indication, while the EMA approved larotrectinib and entrectinib for a tissue-agnostic indication. There were differences in analysis sets (integrated vs. non-integrated), submission dates and requests for data updates between agencies. All agencies had outstanding issues that needed to be addressed in the post-market setting. For pembrolizumab, larotrectinib and entrectinib, the number of imposed PMMs varied between one and eight, with the FDA requesting the most PMMs compared to the other two agencies. All agencies requested at least one PMM per approval to address the remaining uncertainties related to the tissue-agnostic indication. The FDA and EMA requested data from ongoing and proposed trials, while the PMDA requested data from use-result surveys. Confirmation of benefit in the post-marketing setting is an important aspect of tissue-agnostic approvals, regardless of agency. Nonetheless, each approach to confirm benefit has its inherent limitations. Post-marketing data will be essential for the regulatory and clinical decisions-making of medicinal products with a tissue-agnostic indication.

EMA Review of Acalabrutinib for the Treatment of Adult Patients with Chronic Lymphocytic Leukemia.

On November 5, 2020, a marketing authorization valid through the European Union (EU) was issued for acalabrutinib monotherapy or acalabrutinib in combination with obinutuzumab (AcalaObi) in adult patients with treatment-naïve (TN) chronic lymphocytic leukemia (CLL) and also for acalabrutinib monotherapy in adult patients with relapsed or refractory (RR) CLL. Acalabrutinib inhibits the Bruton tyrosine kinase, which plays a significant role in the proliferation and survival of the disease. Acalabrutinib was evaluated in two phase III multicenter randomized trials. The first trial (ACE-CL-007) randomly allocated acalabrutinib versus AcalaObi versus chlorambucil plus obinutuzumab (ChlObi) to elderly/unfit patients with TN CLL. The progression-free survival (PFS), as assessed by an independent review committee, was superior for both the AcalaObi (hazard ratio [HR], 0.1; 95% confidence interval [CI], 0.06-0.17) and acalabrutinib (HR, 0.2; 95% CI, 0.13-0.3) arms compared with the ChlObi arm. The second trial (ACE-CL-309) randomly allocated acalabrutinib versus rituximab plus idelalisib or bendamustine to adult patients with RR CLL. Also in this trial, the PFS was significantly longer in the acalabrutinib arm (HR, 0.31; 95% CI, 0.20-0.49). Adverse events for patients receiving acalabrutinib varied across trials, but the most frequent were generally headache, diarrhea, neutropenia, nausea, and infections. The scientific review concluded that the benefit-risk ratio of acalabrutinib was positive for both indications. This article summarizes the scientific review of the application leading to regulatory approval in the EU. IMPLICATIONS FOR PRACTICE: Acalabrutinib was approved in the European Union for the treatment of adult patients with chronic lymphocytic leukemia who have not received treatment before and for those who have received therapy but whose disease did not respond or relapsed afterward. Acalabrutinib resulted in a clinically meaningful and significant lengthening of the time from treatment initiation to further disease relapse or patient's death compared with standard therapy. The overall safety profile was considered acceptable, and the benefit-risk ratio was determined to be positive.

EMA Recommendation for the Pediatric Indications of Plerixafor (Mozobil) to Enhance Mobilization of Hematopoietic Stem Cells for Collection and Subsequent Autologous Transplantation in Children with Lymphoma or Malignant Solid Tumors.

On March 28, 2019, the Committee for Medicinal Products for Human Use adopted a positive opinion recommending the marketing authorization for the medicinal product plerixafor. The marketing authorization holder for this medicinal product is Genzyme Europe B.Th. The adoption was for an extension of the existing adult indication in combination with granulocyte colony-stimulating factor (G-CSF) to pediatric patients (aged 1 year to <18 years) to enhance mobilization of hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in children with lymphoma or solid malignant tumors. This treatment is indicated either preemptively, when circulating stem cell count on the predicted day of collection after adequate mobilization with G-CSF (with or without chemotherapy) is expected to be insufficient with regard to desired hematopoietic stem cells yield, or in children who previously failed to collect sufficient hematopoietic stem cells. The efficacy and safety of plerixafor were evaluated in an open label, multicenter, phase I/II, dose-ranging, and randomized controlled study (DFI12860) in pediatric patients with solid tumors, including neuroblastoma, sarcoma, Ewing sarcoma, or lymphoma, who were eligible for autologous hematopoietic stem cell transplantation. Forty-five patients (aged 1 year to <18 years) were randomized, 2:1, using 0.24 mg/kg of plerixafor plus standard mobilization (G-CSF with or without chemotherapy) versus control (standard mobilization alone). The primary analysis showed that 80% of patients in the plerixafor arm experienced at least a doubling of the peripheral blood (PB) CD34+ count, observed from the morning of the day preceding the first planned apheresis to the morning prior to apheresis, versus 28.6% of patients in the control arm (p = .0019). The median increase in PB CD34+ cell counts from baseline to the day of apheresis was 3.2-fold in the plerixafor arm versus by 1.4-fold in the control arm. The observed safety profile in the pediatric population was consistent with that in adults, with adverse events mainly related to injection site reactions, hypokalemia, and increased blood bicarbonate. Importantly, plerixafor exposure did not seem to negatively affect transplant efficiency. This article summarizes the scientific review of the application leading to regulatory approval in the European Union. IMPLICATIONS FOR PRACTICE: This review of the marketing authorization of plerixafor will raise awareness of pediatric indication granted for this medicinal product.

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells.

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-ß (TGF-ß) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.

Nucleophosmin1 is a negative regulator of the small GTPase Rac1.

The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2. Here, we report that Rac1 interacts through its C-terminus with nucleophosmin1 (NPM1), a multifunctional nucleo-cytoplasmic shuttling protein with oncogenic properties. We show that Rac1 controls NPM1 subcellular localization. In cells expressing active Rac1, NPM1 translocates from the nucleus to the cytoplasm. In addition, Rac1 regulates the localization of the phosphorylated pool of NPM1 as this pool translocated from the nucleus to the cytosol in cells expressing activated Rac1. Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading. In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1.

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3.

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34⁺ HSPCs. Human CD34⁺ HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34⁺ HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34⁺ HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34⁺ HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34⁺ HSPCs.

Regulation of CXCR4 conformation by the small GTPase Rac1: implications for HIV infection.

The chemokine receptor CXCR4 is a critical regulator of cell migration and serves as a coreceptor for HIV-1. The chemokine stromal cell derived factor-1, also known as CXCL12, binds to CXCR4 and exerts its biologic functions partly through the small guanosine triphosphate hydrolase (GTPase) Rac1 (ras-related C3 botulinum toxin substrate 1). We show in different cell types, including CD34(+) hematopoietic stem and progenitor cells, that inhibition of Rac1 causes a reversible conformational change in CXCR4, but not in the related receptors CXCR7 or CCR5. Biochemical experiments showed that Rac1 associates with CXCR4. The conformational change of CXCR4 on Rac1 inhibition blocked receptor internalization and impaired CXCL12-induced Gα(i) protein activation. Importantly, we found that the conformation adopted by CXCR4 after Rac1 inhibition prevents HIV-1 infection of both the U87-CD4-CXCR4 cell line and of primary peripheral blood mononuclear cells. In conclusion, our data show that Rac1 activity is required to maintain CXCR4 in the responsive conformation that allows receptor signaling and facilitates HIV-1 infection; this implies that Rac1 positively regulates CXCR4 function and identifies the Rac1-CXCR4 axis as a new target for preventing HIV-1 infection.

The chemorepellent Slit3 promotes monocyte migration.

Directional migration is an essential step for monocytes to infiltrate sites of inflammation, a process primarily regulated by chemoattractants. Slits are large matrix proteins that are secreted by endothelial cells; they were reported to inhibit the chemoattractant-induced migration of different cell types, including leukocytes. The aim of this study was to determine the effect of Slit3 on primary monocyte migration and to address the underlying mechanisms. We show that Roundabout (Robo)1, one of the Robo receptors that recognize Slit3, is the only Robo homolog expressed by CD14(+) monocytes. Interestingly, we found that stimulation with Slit3 increased the spontaneous and chemoattractant-induced migration of primary monocytes in vitro and increased the myeloid cell recruitment during peritoneal inflammation in vivo. In addition, Slit3 did not seem to act as a chemoattractant itself; it promoted directed migration triggered by chemoattractants, such as CXCL12, by inducing a chemokinetic effect. We further show that Slit3 prevented monocyte spreading and induced rounding of spread monocytes without affecting monocyte adhesion. Stimulation with Slit3 was not associated with changes in the levels of phosphorylated p38, p42/p44, or Src, known regulators of monocyte migration, but it directly acts on molecular pathways involved in basal leukocyte migration by activating RhoA. These findings show an unexpected response of monocytes to Slit3 and add insights into the possible role of Slit proteins during inflammatory cell recruitment.

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis.

INTRODUCTION: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. RESULTS: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor alpha, interferon gamma and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. CONCLUSIONS: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.

Rho GTPases in hematopoietic cells.

The ubiquitous Rho GTPases are instrumental in the organization of the actin cytoskeleton, but also for the control of gene expression. Here we review the role of the major members of this family, i.e., RhoA, Rac1, Rac2, and Cdc42, and their intracellular signaling in hematopoietic cells. Although these proteins have been classically implicated in chemotaxis, there are now clear indications on how differential signaling toward other, more specific functions, such as phagocytosis or the production of reactive oxygen species, is regulated by relatively small differences in primary sequence. The identification of mutations in these GTPases or their regulators has provided novel insights in their function as well as their relevance for the development of hematological diseases.

Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes.

Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to microvasculature to mediate immune surveillance. Resorption of microvilli is an early morphological alteration induced by chemokines that facilitates lymphocyte emigration. However, the antecedent molecular mechanisms remain largely undefined. We demonstrate that Rac1 plays a fundamental role in chemokine-induced microvillar breakdown in human T lymphocytes. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galphai. Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal "Trojan" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine.

The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity.

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.

Somatostatin is a selective chemoattractant for primitive (CD34(+)) hematopoietic progenitor cells.

OBJECTIVE: Somatostatin (SST) is a regulatory peptide with a wide variety of activities in different tissues. SST activates G(alpha i)-protein-coupled receptors of a family comprising five members (SSTR1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of activities of SST is incompletely defined. Here, we examined the role of SST and its receptors in hematopoiesis. MATERIALS AND METHODS: SSTR expression on human and mouse hematopoietic cells was analyzed by flow cytometry and reverse transcriptase polymerase chain reaction. The effects of SST on cell migration were measured in transwell assays. Using selective inhibitors, signaling mechanisms involved in SSTR2-mediated migration were studied in 32D cell transfectants expressing SSTR2. RESULTS: Human hematopoietic cells exclusively expressed SSTR2, whereas mouse bone marrow cells expressed SSTR2 and SSTR4. SSTR levels were high on primitive (CD34(+), Lin(-)) but low or absent on more mature (CD34(-), Lin(+)) cell types. Both SST and its analog octreotide acted as chemoattractants for primitive hematopoietic cells. Despite the presence of SSTR4, bone marrow cells from SSTR2-deficient mice failed to migrate toward SST gradients, suggesting that SSTR2 and SSTR4 are functionally different in this respect. SST activated phosphatidylinositol 3-kinase and the MAP kinases Erk1/2 and p38 in 32D[SSTR2] cells. While chemical inhibitors of these kinases had some effect, SST-induced migration was most strongly affected by blocking G(alpha i) activity or by elevating intracellular cAMP levels. CONCLUSIONS: Somatostatin acts as a selective chemoattractant for immature hematopoietic cells via activation of multiple intracellular pathways.