Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Physiological restraint of Bak by Bcl-xL is essential for cell survival.

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

The rise of apoptosis: targeting apoptosis in hematologic malignancies.

Dysregulation of the B-cell leukemia/lymphoma-2 (BCL-2) family of proteins of the intrinsic apoptotic pathway is fundamental to the pathophysiology of many hematologic malignancies. The BCL-2 family consists of regulatory proteins that either induce apoptosis (proapoptotic) or inhibit it (prosurvival). BCL-2, myeloid cell leukemia-1, and B-cell lymphoma-extra large are prosurvival proteins that are prime targets for anticancer therapy, and molecules targeting each are in various stages of preclinical and clinical development. The US Food and Drug Administration (FDA)-approved BCL-2 inhibitor venetoclax was first proven to be highly effective in chronic lymphocytic leukemia and some B-cell non-Hodgkin lymphoma subtypes. Subsequently, venetoclax was found to be active clinically against a diverse array of hematologic malignancies including multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, and others. Here, we give a brief introduction to BCL-2 family biology and the mechanism of action of BCL-2 Homology 3 (BH3) mimetics, and provide an overview of the clinical data for therapeutically targeting prosurvival proteins in hematologic malignancies, with a focus on BCL-2 inhibition. To prioritize novel agent combinations and predict responders, we discuss the utility of functional assays such as BH3 profiling. Finally, we provide a perspective on how therapies targeting BCL-2 family proteins may be optimally implemented into future therapeutic regimens for hematologic malignancies.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells.

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

Antagonism between MCL-1 and PUMA governs stem/progenitor cell survival during hematopoietic recovery from stress.

Understanding the critical factors that govern recovery of the hematopoietic system from stress, such as during anticancer therapy and bone marrow transplantation, is of clinical significance. We investigated the importance of the prosurvival proteins myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma-extra large (BCL-XL) in stem/progenitor cell survival and fitness during hematopoietic recovery from stress. Loss of a single Mcl-1 allele, which reduced MCL-1 protein levels, severely compromised hematopoietic recovery from myeloablative challenge and following bone marrow transplantation, whereas BCL-XL was dispensable in both contexts. We identified inhibition of proapoptotic p53 upregulated modulator of apoptosis (PUMA) as the key role of MCL-1 in both settings, with Mcl-1(+/-);Puma(-/-) mice completely protected from the deleterious effects of loss of 1 Mcl-1 allele. These results reveal the molecular mechanisms that govern cell survival during hematopoietic recovery from stress.

MOZ regulates B-cell progenitors and, consequently, Moz haploinsufficiency dramatically retards MYC-induced lymphoma development.

The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZ gene to CBP, p300, NCOA3, or TIF2 in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eµ-Myc model of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Moz increased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Moz haploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1 translocations in leukemia. We demonstrate that MOZ localizes to the Meis1 locus in pre-B-cells and maintains Meis1 expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.

Loss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1).

Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.

MCL-1 but not BCL-XL is critical for the development and sustained expansion of thymic lymphoma in p53-deficient mice.

Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.

XIAP discriminates between type I and type II FAS-induced apoptosis.

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis.

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Endogenous Bcl-xL is essential for Myc-driven lymphomagenesis in mice.

Impaired apoptosis is a cancer hallmark, and some types of lymphomas and other cancers harbor mutations that directly affect key cell death regulators, such as Bcl-2 family members. However, because the majority of tumors seem to lack such mutations, we are examining the hypothesis that tumorigenesis can be sustained at least initially by the normal expression of specific endogenous pro-survival Bcl-2 family members. We previously demonstrated that the lymphomagenesis in Εµ-myc transgenic mice, which constitutively overexpress the c-Myc oncoprotein in B-lymphoid cells and develop pre-B and B-cell lymphomas, does not require endogenous Bcl-2. In striking contrast, we report here that loss in these mice of its close relative Bcl-x(L) attenuated the pre-neoplastic expansion of pro-B and pre-B cells otherwise driven by c-Myc overexpression, sensitized these cells to apoptosis and ablated lymphoma formation. Remarkably, even loss of a single bcl-x allele delayed the lymphomagenesis. These findings identify Bcl-x(L) as a prerequisite for the emergence of c-Myc-driven pre-B/B lymphoma and suggest that BH3 mimetic drugs may provide a prophylactic strategy for c-Myc-driven tumors.

BH3 mimetic drugs cooperate with Temozolomide, JQ1 and inducers of ferroptosis in killing glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

Combined reduction in the expression of MCL-1 and BCL-2 reduces organismal size in mice.

The intrinsic apoptotic pathway is controlled by the BCL-2 family of proteins, which exhibit either a pro-death or pro-survival function. Gene knockout studies revealed that different pro-survival BCL-2 proteins are critical for the survival of distinct cell types, although overlapping functions amongst such proteins have also been identified. In the process of studying mice lacking single alleles of Mcl-1 (Mcl-1+/-), Bcl-2 (Bcl-2+/-), or both in combination (Mcl-1+/-Bcl-2+/-), we observed that Mcl-1+/-Bcl-2+/- mice weighed less when compared with their wild-type littermates as they aged. Body composition analysis demonstrated that while fat mass was similar to wild-type controls, lean mass was significantly reduced in Mcl-1+/-, Bcl-2+/-, and, most strikingly in Mcl-1+/-Bcl-2+/- mice. The weights of several tissues including the heart, tibialis anterior, and kidney were likewise reduced in Mcl-1+/-Bcl-2+/- mice. When lean mass and specific tissue weights were expressed relative to body weight, these differences were no longer significant, indicating that that Mcl-1+/-Bcl-2+/- mice, and to a lesser extent Mcl-1+/- and Bcl-2+/- mice, are smaller than their wild-type counterparts. Consistently, the anal-naso length was reduced in Mcl-1+/-Bcl-2+/- mice. While minor reductions in size were observed in female Mcl-1+/-Bcl-2+/- mice, these effects were most prominent in males. Notably, Mcl-1+/-Bcl-2+/- males had markedly smaller testes even after accounting for differences in body weight. Collectively, these data reveal that combined loss of a single allele of Mcl-1 and Bcl-2, while not overtly impairing organismal development, leads to a reduction in animal size.

Synergy between EphA2-ILs-DTXp, a Novel EphA2-Targeted Nanoliposomal Taxane, and PD-1 Inhibitors in Preclinical Tumor Models.

Combinations of chemotherapy with immunotherapy have seen recent clinical success, including two approvals of anti-PD-1/L1 agents in combination with taxane-based chemotherapy in non-small cell lung cancer and triple-negative breast cancer. Here, we present a study on the combination activity and mechanistic rationale of a novel EphA2-targeted liposomal taxane (EphA2-ILs-DTXp) and anti-PD-1. This combination was highly active in mouse syngeneic tumor models, with complete responses observed in 3 of 5 models. In the EMT-6 tumor model, combination of EphA2-ILs-DTXp with anti-PD-1 resulted in a 60% complete response rate, with durable responses that were resistant to rechallenge. These responses were not observed in the absence of CD8+ T cells. Characterization of the immune infiltrates in EMT-6 tumors reveals increased CD8+ T cells, increased CD8+ IFNγ+ CTLs, and an increased CD8/regulatory T-cell (Treg) ratio. These immunomodulatory effects were not observed in mice treated with a combination of docetaxel and anti-PD-1. Pharmacokinetic analysis revealed that the AUC of docetaxel was increased 15 times, from 52.1 to 785 ng/mL/hour, when delivered by EphA2-ILs-DTXp. A dose reduction study of EphA2-ILs-DTXp showed a dose-response relationship for both tumor growth inhibition and the CD8/Treg ratio. Our data indicate that synergism between docetaxel and anti-PD-1 is achievable with nanoliposomal delivery.

Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer.

Radiation therapy induces immunogenic cell death, which can theoretically stimulate T cell priming and induction of tumor-specific memory T cell responses, serving as an in situ vaccine. In practice, this abscopal effect is rarely observed. We use two mouse models of pancreatic cancer to show that a single dose of stereotactic body radiation therapy (SBRT) synergizes with intratumoral injection of agonistic anti-CD40, resulting in regression of non-treated contralateral tumors and formation of long-term immunologic memory. Long-term survival was not observed when mice received multiple fractions of SBRT, or when TGFß blockade was combined with SBRT. SBRT and anti-CD40 was so effective at augmenting T cell priming, that memory CD8 T cell responses to both tumor and self-antigens were induced, resulting in vitiligo in long-term survivors.

Subtle Changes in the Levels of BCL-2 Proteins Cause Severe Craniofacial Abnormalities.

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1+/-;Bcl-x+/- mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members.