SUMMARY
MCL-1 is a pro-survival BCL-2 protein required for the sustained growth of many cancers. Recently a highly specific MCL-1-inhibitor, S63845, showing 6-fold higher affinity to human compared to mouse MCL-1 has been described. To accurately test efficacy and tolerability of this BH3 mimetic drug in pre-clinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse in which MCL-1 was replaced with its human homologue. HuMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to MCL-1 inhibition. Importantly, non-transformed cells and lymphomas from huMcl-1;Eμ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eμ-Myc lymphoma cells are transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide leads to long-term remission in ~60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMCL-1 mouse model to test MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.
INTRODUCTION
Apoptosis is a form of programmed cell death required for the removal of unwanted and potentially dangerous (e.g. infected) cells in multicellular organisms (Green and Llambi, 2015). Deregulation of the intrinsic apoptotic pathway can lead to accumulation of cells that should normally be deleted, including those with DNA lesions. The abnormal proliferation of damaged and mutated “undead” cells, can lead to tumour development. Cell death hinges on the balance between the pro-survival and pro-apoptotic members of the BCL-2 family of proteins. Defined by the presence of at least one of four BCL-2 homology (BH) domains, this family is further divided into the pro-survival members (BCL-2, BCL-XL, BCL-W, MCL-1 and BCL2A1/BFL-1), the BH3-only pro-apoptotic proteins (such as BIM and PUMA) and the multi BH domain pro-apoptotic effectors (BAK, BAX and BOK). Diverse stress stimuli cause increased expression of BH3-only family members to initiate apoptosis, either indirectly by binding to the pro-survival BCL-2 proteins, thereby unleashing the pro-apoptotic effectors BAK and BAX, or by binding BAK and BAX directly. Activation of BAK and BAX leads to their oligomerization on the outer mitochondrial membrane, perforating its surface, thereby releasing cytochrome-c and other apoptogenic factors. This induces a cascade of signalling events driven by the caspases that causes demolition of the cell.
Cancer cells can subvert the apoptotic machinery by either up-regulation of pro-survival proteins or down-regulation of pro-apoptotic proteins, thereby maximizing their growth potential (Hanahan and Weinberg, 2011). MCL-1 is important for the sustained growth of many cancers. The MCL-1 locus is amplified in numerous human tumor types (Beroukhim et al., 2010) and functional studies in mouse models have showed that MCL-1 is essential for the sustained growth of many cancers, including multiple myeloma (Gong et al., 2017), T cell lymphomas (Grabow et al., 2014; Spinner et al., 2016), MYC- (Kelly et al., 2014) or BCR-ABL-driven (Koss et al., 2013) pre-B/ B cell lymphomas, acute myeloid leukaemia (Glaser et al., 2012) as well as some subtypes of solid tumours (Xiao et al., 2015; Zhang et al., 2011). Direct targeting of pro-survival BCL-2 family members by so-called ‘BH3 mimetics’ is a successful approach in cancer therapy. The BCL-2 inhibitor Venetoclax is highly efficacious for the treatment of relapsed/refractory Chronic Lymphocytic Leukemia (CLL) (Roberts et al., 2016; Stilgenbauer et al., 2016). A clinically relevant specific and potent inhibitor of MCL-1, called S63845 (Kotschy et al., 2016), has been shown to be efficacious as a single agent in several pre-clinical models of haematological malignancies and in combination with oncogenic kinase inhibitors in certain lung, skin as well as breast cancer derived cell lines and patient-derived xenograft models (Kotschy et al., 2016; Merino et al., 2017).
The clinical application of such an MCL-1 inhibitor has been of great concern given the important role MCL-1 plays in many normal tissues. MCL-1 is widely expressed (Kozopas et al., 1993), and essential for embryonic development with homozygous loss of Mcl-1 in mice resulting in failure to implant at the blastocyst stage (Rinkenberger et al., 2000). Furthermore, conditional gene knockout studies have shown that MCL-1 plays a vital role in the survival of cardiomyocytes (Thomas et al., 2013; Wang et al., 2013), hematopoietic stem cells (Opferman et al., 2005), developing and mature lymphocytes (Dzhagalov et al., 2008; Opferman et al., 2003; Peperzak et al., 2013; Vikstrom et al., 2010) and in the maintenance of oocytes in the ovarian reserve (Omari et al., 2015). In spite of the many important roles MCL-1 plays, very little toxicity was observed when healthy mice were treated in vivo with the MCL-1 inhibitor S63845 at a dose that ablates mouse lymphoma cells (Kotschy et al., 2016). One caveat of these results is that S63845 has a ~6-fold higher affinity for the human protein in comparison to murine MCL-1 and therefore it is possible that the mouse models utilized were not sensitive enough to reveal all potential on-target toxicities. It was therefore pivotal to test more accurately the action of S63845 in a pre-clinical model in which we had humanized the Mcl-1 locus (huMcl-1) by replacing the native coding region of the murine Mcl-1 locus with the human MCL-1 coding sequence. These mice are healthy and fertile, and the intrinsic apoptotic pathway is intact in their cells, demonstrating that the human MCL-1 protein can functionally fully replace the murine protein. As predicted, cells from huMcl-1 mice were more sensitive to S63845 compared to wild-type cells, but not other cytotoxic agents that induce killing by the apoptotic pathway. In line with these results the maximum tolerated dose of S63845 was lower in the huMcl-1 mice compared to their wild-type counterparts. However, it was still possible to cure huMcl-1 mice of MYC-driven lymphomas expressing human MCL-1 by treatment with S63845, either alone or at lower doses together with cyclophosphamide, without causing overt damage to healthy tissues. These findings demonstrate the utility of the huMcl-1 model to accurately determine the efficacy and tolerability of S63845 and potentially other MCL-1 inhibitory drugs in pre-clinical models of disease.
RESULTS
Generation of the humanised Mcl-1 mouse model
The humanized Mcl-1 (huMcl-1) mouse model was generated by standard gene targeting in C57BL/6 embryonic stem (ES) cells. Expression of the human MCL-1 protein under the control of the murine Mcl-1 promoter and regulatory regions was achieved by substitution of the exons and interspersing introns of the mouse Mcl-1 locus with the human MCL-1 exon/intron sequences, while maintaining the flanking 5’ and 3’ untranslated region of the genomic locus of the mouse (Fig. 1A). Homozygous loss of Mcl-1 causes embryonic lethality prior to embryonic-day 4 in mice (Rinkenberger et al., 2000). Furthermore, changes in the 5’ untranslated region of the murine Mcl-1 locus can lead to infertility in homozygous male Mcl-1 floxed mice (Okamoto et al., 2014). Importantly, we found that the humanization of MCL-1 in mice had no impact on fertility or embryonic development, with the expected Mendelian ratios and sex distribution observed in intercrosses of Mcl-1hu/wt mice (Fig. 1B). Western blot analysis of thymocytes from huMcl-1 and wild-type mice showed efficient expression of the human MCL-1 protein, as evidenced by the predicted size difference between the mouse and human MCL-1 proteins (human: 38 kD, mouse: 35 kD, Fig. 1C). A cohort of huMcl-1 mice was aged for >600 days without showing any obvious signs of defects.
Humanised Mcl-1 mice have normal hematopoietic cell subset distribution
MCL-1 is crucial for the survival of many developing and mature cell subsets of the hematopoietic system in mice (Dzhagalov et al., 2008; Huntington et al., 2007; Opferman et al., 2005; Opferman et al., 2003; Peperzak et al., 2013; Vikstrom et al., 2010). FACS analysis of the bone marrow revealed no differences in the frequencies and numbers of pro-B/pre-B (B220loIgM−), immature B (B220loIgM+), transitional B (B220midIgMhi) or mature B (B220hiIgMlo) cell subsets between huMcl-1 mice and wild-type controls (representative FACS plots shown; Fig. 1D). There were also no abnormalities in IgM+ or IgD+ peripheral B lymphocytes in the huMcl-1 mice (Fig. 1E). The frequencies and numbers of the different stages of thymocyte development, as determined by staining for CD4 and CD8, were also comparable between huMcl-1 mice and wild-type controls (representative FACS plot shown; Fig. 1F). Moreover, huMcl-1 mice had normal frequencies and numbers of mature CD4+ and CD8+ T lymphocytes in the spleen (Fig. 1G), with normal distributions of naïve (CD62L+CD44−), effector memory (CD62L−CD44+) and central memory (CD62L+CD44+) CD4+ T cells (Fig. S1A). Finally, the huMcl-1 mice had normal frequencies and numbers of macrophage/monocyte (MAC-1+GR-1lo) and neutrophil populations (MAC-1+GR-1hi) in the bone marrow and spleen (Fig. S1B).
Human MCL-1 protein can functionally replace mouse MCL-1 within the apoptotic machinery
Since it is possible that other BCL-2 proteins (pro-apoptotic or pro-survival) are differentially expressed to compensate for the expression of huMCL-1 instead of the mouse MCL-1 protein in vivo, we determined the levels of these proteins by Western blotting. Thymocytes and splenocytes from huMcl-1 mice showed no difference in the protein levels of the pro-apoptotic BIM and PUMA or pro-survival BCL-2, BCL-XL or A1 (Representative Western blot Fig. 2A, multiple blots quantified in Fig. 2B). The affinity for MCL-1 antibodies to the mouse versus human protein is not known, thus we could not compare the relative protein expression by Western blot. To test whether the huMCL-1 protein expressed in mouse cells is able to interact with the endogenous mouse pro-apoptotic BCL-2 relatives, we performed co-immunoprecipitation assays on thymocyte extracts. This revealed that the huMCL-1 protein expressed in mouse cells is capable of binding to mouse BIM and BAK in a similar manner as mouse MCL-1 (Fig. 2C). To assess the functionality of the huMCL-1 protein, we investigated the response of cells from the huMcl-1 mice to a diverse range of cytotoxic stimuli in vitro. Importantly, no abnormalities were observed in the survival of thymocytes or B cells from the huMcl-1 mice (Fig. 2D). As predicted, thymocytes and B cells from the huMcl-1 mice were more sensitive to the MCL-1 inhibitor S63845 compared to those from wild-type mice (Fig. 2E). While the increased sensitivity to S63845 became only obvious at higher (200 nM, 1 μM) doses in thymocytes, more striking differences were observed in B cells after 6 hours of treatment with low (40 nM) or high (1 μM) doses of the MCL-1 inhibitor (Fig. 2E, 24h Fig. S2A).
Next, we wanted to test the ability of huMCL-1 protein expressed in mouse cells to function within a whole animal. MCL-1 is required for the maintenance of hematopoietic stem/progenitor cells (Opferman et al., 2005) and for emergency hematopoiesis following myeloablative chemotherapy (Delbridge et al., 2015). The latter was demonstrated when Mcl-1wt/- mice were found to be compromised in their recovery from 5-fluorouracil (5-FU) treatment. We treated huMcl-1, Mcl-1wt/- (as a control) and wild-type mice with a single dose of 5-FU (150 mg/kg body weight) and monitored for hematopoietic recovery over a period of 21 days. As reported (Delbridge et al., 2015), most Mcl-1wt/- mice did not recover from 5-FU treatment, whereas huMcl-1 mice recovered as well as wild-type controls (Fig 2F). Collectively, these findings demonstrate that huMCL-1 is functional and the apoptotic machinery is intact in cells from the huMcl-1 mice, both in vitro and in vivo. Furthermore, as predicted, these cells show increased sensitivity to the MCL-1 inhibitor S63845.
Determining the maximum tolerated dose of S63845 in humanized Mcl-1 mice
Given the binding affinity of S63845 is ~6-fold higher for human MCL-1 protein compared to mouse MCL-1, we determined the maximum tolerated dose (MTD) of S63845 in the huMcl-1 mice. Treatment of huMcl-1 mice on 5 consecutive days intravenously (i.v.) with doses ranging from 5 to 25 mg/kg body weight established the MTD of S63845 at 12.5 mg/kg (Fig 3A). At 15 mg/kg S63845, 1 out of 4 mice did not survive while none of the mice could tolerate the drug at 25 mg/kg. The tolerability of the drug was considerably higher in wild-type mice, which all survived the 25 mg/kg dosing schedule (Fig.3A) and have previously been shown to have an MTD of 40 mg/kg (Kotschy et al., 2016). This demonstrates that the huMcl-1 mice are more sensitive to the MCL-1 inhibitor S63845.
S63845 exerts no enduring toxicity in the huMcl-1 mice at the MTD
There have been many reports on the importance of MCL-1 in several vital tissues (Dzhagalov et al., 2008; Omari et al., 2015; Opferman et al., 2005; Opferman et al., 2003; Peperzak et al., 2013; Thomas et al., 2013; Vikstrom et al., 2010; Wang et al., 2013) raising the question whether an MCL-1 inhibitor would find its therapeutic use in the clinic. Previously it was shown that S63845 exerts very little toxic effects in mice expressing mouse MCL-1 (Kotschy et al., 2016), but given the higher affinity of this drug for human MCL-1 we revisited this question by treating huMcl-1 (and wild-type controls) mice for 5 consecutive days with either vehicle or 12.5 mg/kg S63845 and testing for acute impact and recovery 3 or 17 days post treatment, respectively. In the huMcl-1 mice, the pro-B/pre-B (B220loIgM−), immature B (B220loIgM+), transitional B (B220midIgMhi) and mature B (B220hiIgMlo) B cells in the bone marrow were significantly reduced at 3 days but had mostly recovered at 17 days post treatment (representative FACS plots Fig. 3B; total cell numbers Fig. 3C). Similar observations were made for B cells in the blood (Fig. S3A) and spleen (Fig. 3D). Despite the reduction in splenic B cells, an increase in cellularity was noted in the spleens of all huMcl-1 mice treated with S63845 3 days after the treatment, but these values returned to normal at 17 days post treatment (Fig. S3B). FACS analysis revealed that the increased splenic cellularity was due to an increase in EryA (Lineage−Ter119hiCD71+FSC-Ahi) and EryB (Lineage-Ter119hiCD71+FSC-Alo) erythrocyte progenitor populations, with the more mature EryC (Lineage−Ter119hiCD71−FSC-Ato) remaining stable throughout the experiment (representative FACS plots Fig. S3C; total cell numbers Fig. S3D). Moreover, the RBC counts in the peripheral blood showed a modest reduction at day 3 post treatment, but again these numbers were completely recovered at day 17 (Fig.S3E). No significant changes were observed in the LSK (Lineage−SCA-1+c-KIT+, Fig. 3E), or neutrophil (MAC-1+GR-1hi) populations in the bone marrow (Fig. 3E) or spleen (Fig. S3F) of the S63845 treated huMcl-1 mice. Moreover, there were no changes in the numbers of mature CD4+ and CD8+ T cells (Fig 3F), immature double negative (DN1-4, Lineage−TCRb− CD4−CD8−) and double positive (DP, CD4+CD8+) thymocytes (Fig. S3G) or mature T cells (TCRß+) in the spleen of S63845 treated huMcl-1 mice (Fig S3G). Histological analysis revealed an increased size and loss of normal architecture of the spleen, which complements the FACS data (Fig. 3G). Histological analysis of major organs revealed no damage in response to S63845 (Fig. 3F). These data indicate that S63845 treatment can be tolerated in huMcl-1 mice, with only a transient reduction of certain hematopoietic cell subsets.
Eμ-Myc cell lines expressing huMcl-1 are more sensitive to S63845
In order to test whether the sensitivity towards MCL-1 inhibition with S63845 could be increased not only in healthy but also malignant cells, we crossed the huMcl-1 mice with the Eμ-Myc transgenic animals. While the latency and tumour phenotype did not differ between mouse or human MCL-1 expressing Eμ-Myc mice (Fig. 4A-B), the in vitro sensitivity of cell lines generated from sick mice increased by 6-fold in the Eμ-Myc;huMcl-1 lymphoma cells towards MCL-1 inhibition with S63845 compared to control Eμ-Myc lymphoma cells expressing mouse MCL-1 protein (25nM or 160nM respectively, Fig. 4C-D).
Regression of huMcl-1;Eμ-Myc lymphomas in vivo with S36845, either alone or in combination with cyclophosphamide
To test the sensitivity of Eμ-Myc lymphomas expressing human MCL-1 to S63845 in vivo, we transplanted huMcl-1;Eμ-Myc lymphoma cell lines into huMcl-1;Ly5.1 recipient mice (i.e. both lymphoma and normal cells expressed huMCL-1). After 3 days, mice were treated for 5 consecutive days with vehicle or 12.5 mg/kg S63845 and monitored for signs of sickness. 60% of mice were cured at this dose (Fig. 4E, survival curves of individual cell lines; Fig. S4A). Next, we aimed to improve tumour free survival in mice transplanted with huMcl-1;Eμ-Myc lymphoma cell lines. To this end, huMcl-1;Eμ-Myc lymphoma cell lines were transplanted into huMcl-1;Ly5.1 recipient mice and 2 days later treated with vehicle or a low dose of cyclophosphamide (CP, 50 mg/kg). Three days later mice were treated for 5 consecutive days with a low dose of S63845 (7.5 mg/kg). Treatment with 50mg/kg CP or 7.5mg/kg S63845 by themselves resulted in ~50% or ~25% tumor free survival, respectively. Excitingly, only one mouse became sick with lymphoma in the combination treatment increasing the efficacy of S63845 thereby increasing the tumor free survival to almost 100% (Fig. 4F, individual cell lines: Fig. S4B). For cell lines that did previously regress at 12.5mg/kg bodyweight S63845 (hME160, hME184, hME273; Fig. S4A), cancer free survival was also achieved with as little as 3.75mg/kg bodyweight S63845 in combination with CP (Fig 4G). These findings, in a highly relevant mouse model in which both lymphoma and normal cells express huMCL-1, clearly demonstrate that there is a therapeutic window for S63845, and that its efficacy can be enhanced by combining treatment with cyclophosphamide.
DISCUSSION
Here we describe a novel humanized Mcl-1 mouse model, in which human MCL-1 protein is expressed under the control of the mouse Mcl-1 regulatory regions. Normal embryonic development and fertility of these mice is noteworthy given that even small changes to the Mcl-1 locus or loss of only one allele bear consequences in adult mice (Okamoto et al., 2014), with homozygous loss of Mcl-1 being embryonic lethal very early in embryonic development (Rinkenberger et al., 2000). Moreover, the intrinsic apoptotic pathway remains intact in cells from the huMcl-1 mice with no compensatory changes in the levels of other BCL-2 family members detected (Fig. 2). This is critical because changes in expression of the BCL-2 family member proteins can compensate for the loss of another. For example, tumor cells can acquire resistance to inhibitors of BCL-2, BCL-XL and BCL-W (e.g. ABT-737) by up-regulating MCL-1 (Lin et al., 2007). Since the huMcl-1 mice are normal and their cells have an unimpaired apoptotic machinery (confirmed both in vitro and in vivo) they represent an ideal model to predict tolerability and efficacy of MCL-1 inhibitors.
MCL-1 is regarded as an exciting therapeutic target due to its importance for the sustained growth of many cancers, and S63845 has been developed as a highly selective and potent inhibitor. A so far unique feature for BH3 mimetics is that S63845 binds more tightly to the human MCL-1 protein compared to the mouse MCL-1 protein and one could imagine that any newly generated compounds targeting MCL-1 would likely bind in the same region, and therefore, will likely have similar properties. Since previous in vivo experiments have all been conducted with mouse MCL-1 as the target, it was difficult to determine whether the results are truly reflective of the therapeutic potential of S63845 and perhaps more importantly, the safety. Our studies using the huMcl-1 mouse model support the notion that a therapeutic window for MCL-1 inhibitors might be established, with a transient loss of B cells being the most significant side effect observed at doses of S63845 that could halt lymphoma growth in a substantial fraction of mice. These data may be viewed conflicting with previous data showing the importance of MCL-1 in many essential normal cell types, such as cardiomyocytes. However, we believe that the lack of significant side effects is due to the transient action and hence inhibition of MCL-1 by the drug in contrast to the irreversible loss of MCL-1 elicited by genetic deletion. Additionally, it is not yet known whether S63845 is available in all organs throughout the mouse after its administration, which may further explain the differences seen between deletion vs inhibition of MCL-1. Further pharmacokinetic studies must be performed to address this question.
While S63845 appears to be relatively well tolerated as previously shown in mice expressing mouse MCL-1, we have shown that S63845’s tighter binding to human MCL-1 is relevant in vivo with the MTD of S63845 in huMcl-1 mice being ~3 times lower than that of wild-type mice. This suggests that the huMcl-1 mouse model will be useful for future pre-clinical work using S63845 (and other MCL-1 inhibitors) for determining a therapeutic window for treatment of cancers and possibly other diseases in which MCL-1 is important. As a proof of principal, we generated huMcl-1;Eμ-Myc lymphomas and showed that they are more sensitive to S63845, both in vitro and in vivo, than Eμ-Myc lymphomas expressing mouse MCL-1. The observation that huMcl-1 mice could tolerate doses of S63845 that could prevent growth of huMcl-1;Eμ-Myc lymphomas suggests that a therapeutic window of MCL-1 inhibitors may be established in the clinic. While there are already phase I clinical trials being conducted for MCL-1 inhibitors, it is important to generate relevant pre-clinical data in different cancer models using our huMcl-1 mice to help make rational decisions on which malignancies will benefit the most in these early stages of clinical development, and to determine which other anti-cancer agents can cooperate with MCL-1 inhibitors in tumor cell killing and still be tolerable as a combination therapy. Of note, we are currently breeding the huMcl-1 alleles onto the NOD/SCID/common-gamma chain knockout (NSG) background, which will allow the testing of MCL-1 on primary human cancer cells (xenografts) with human MCL-1 expression in the healthy host cells. These highly relevant pre-clinical models will help guide the safe and appropriate use of MCL-1 inhibitors in cancer patients.
AUTHOR CONTRIBUTIONS
The experiments were conceived and designed by MSB, AS, GLK and MJH. Experiments were performed by MSB, CC, GD, LT and GLK. GL contributed expertise and provided reagents. The paper was written by MSB, AS, GLK and MJH with help from the other authors.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
STAR METHODS
Generation of the humanized Mcl-1 mice
Mice bearing the huMcl-1 allele were generated by Taconic Biosciences GmbH, Cologne, Germany. The targeting vector (designed to insert the human MCL-1 coding sequence under the control of the endogenous Mcl-1 promoter, Fig. 1A) was transfected into the TaconicArtemis C57BL/6NTac embryonic stem (ES) cell line. Homologous recombinant clones were isolated using positive (PuroR) and negative (Thymidine kinase - TK) selection. Correctly targeted ES cells were injected into BALB/c blastocysts that were then transferred into pseudo-pregnant NMRI females. Chimeric offspring were selected by coat color for further breeding. The PuroR sequence was flanked by FRT sites to allow subsequent removal of the selection cassette by crossing chimeric mice with the C57BL/6-Tg(CAG-Flpe)2 Arte transgenic mice carrying the FLP recombinase (FLPe), giving rise to the final huMcl-1 allele. Genotyping was performed by PCR to confirm cassette removal, loss of the FLPe transgene, and presence of the wild-type or humanized Mcl-1 alleles (For PCR primers see Supplementary Table 1).
Co-immunoprecipitation and Western blotting
Thymocytes were isolated from Mcl-1wt/wt and Mcl-1hu/hu mice and prepared in lysis buffer (Supplementary Table S2) supplemented with protease inhibitors cOmplete and pepstatin A (Roche and Sigma). Cell lysates were collected and pre-cleared with sepharose beads for 1 h at 4°C with constant agitation. Immunoprecipitation was performed using 2.5 μg monoclonal antibody against MCL-1 (Supplementary Table S2) incubated overnight at 4°C, followed by incubation with protein G sepharose beads for 1 h at 4°C. Immunoprecipitated proteins were eluted by boiling in SDS-PAGE sample buffer for 5 min and analyzed by Western blotting (antibodies listed in Supplementary Table S2). For all other Western blots, cell lysates were prepared in RIPA buffer (Supplementary Table S3) supplemented with complete protease inhibitor (Roche). Protein concentration was determined by Bradford assay using the Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Samples of 15 mg protein were prepared in Laemmli buffer (Supplementary Table S3), boiled for 5 min and size fractionated by gel electrophoresis on NuPAGE 10% Bis-Tris 1.5 mm gels (Life Technologies) in MES buffer and then transferred onto nitrocellulose membranes (Life Technologies) using the iBlot membrane transfer system. Antibody dilution and blocking were performed in 5% skim milk, 0.1% Tween 20 in PBS. For antibodies refer to Supplementary Table S3, in house antibodies (Lang et al., 2014; Okamoto et al., 2014). Luminata Forte Western HRP substrate (Millipore, Billerica, MA, USA) was used for developing and membranes were imaged and analyzed using the ChemiDoc XRS+ machine with ImageLab software (Bio-Rad).
Immunostaining andflow cytometry
Thymus, spleen and bone marrow were harvested and single cell suspensions prepared in PBS (Gibco), 5 mM EDTA (Merck), supplemented with 5% fetal bovine serum (FBS, Sigma-Aldrich) for staining. Monoclonal antibodies (Supplementary Table 4) were obtained from eBioscience, BioLegend or generated at the Walter and Eliza Hall Institute (WEHI) Antibody Facility. Streptavidin-PE (BioLegend) was used to detect biotinylated antibodies. Propidium iodide (PI, 1 μg/mL) was used to exclude dead cells. For steady state analysis (Fig. 1, 1S), Whole organ cell counts were determined by the CASY counter (Schärfe System GmbH)(Fig. 1, S1) or by mixing a known concentration of APC Calibrite beads (Becton Dickinson) with each sample (Fig. 3, 3S). Data were collected using LSR II, LSRFortessa or LSRFortessa X-20 analyzers and examined using FlowJo 10 (Becton Dickson).
Tissue culture and cell viability assays
Eμ-Myc lymphoma cell lines were maintained as previously described (Kelly et al., 2014). For all experiments, viability was determined by resuspending cells in Annexin V binding buffer (0.1 M Hepes (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) containing PI (1 μg/mL) and FITC- or Alexa Fluor 647-conjugated Annexin V (generated in house). Spleen and thymi were harvested and single cell suspensions prepared. To isolate B cells, splenocytes were pelleted and resuspended in 1 mL red cell lysis buffer (156 mM ammonium chloride (BDH), 11.9mM sodium bicarbonate (Merck), EDTA (Sigma)) for 5 min to deplete erythrocytes. Cells were then stained with biotinylated antibodies against CD4, CD8, MAC-1 and GR-1 (Supplementary Table S4) to enrich for B cells by MagniSort Streptavidin Negative Selection Beads (Thermo Fisher), as per the manufacturer’s protocol. Isolated B cells and thymocytes were seeded at 5 × 104 cells/well, in triplicate per condition, in 96 well flat-bottomed plates with either S63845 (8, 200, 1000 nM, gift from Servier), dexamethasone (1 nM), etoposide (1 □g/mL), PMA (10 ng/mL) or ionomycin (1 μg/mL) (Sigma) for the indicated times. The sensitivity of Eμ-Myc lymphoma cell lines to S63845 was determined by seeding 5 × 104 lymphoma cells into 96 well flat-bottomed plates in FMA medium with 5 concentrations of S63845 (1:5 dilutions starting from 1 μM, gift from Servier), in triplicate and incubated at 37°C and 10% CO2 for 24 h. The IC50 values were determined using nonlinear regression algorithms in Prism (GraphPad).
Animals and in vivo drug treatments
The care and use of mice for experimental purposes were carried out in accordance with the requirements set out by the Walter and Eliza Hall Institute (WEHI) Animal Ethics Committee. HuMcl-1 mice are described above; Eμ-Myc (Adams et al., 1985) and Mcl-1wt/- (Vikstrom et al., 2010) mice have been described previously. All mice are kept on a C57BL/6-Ly5.2 background. The huMcl-1 allele was bred onto a C57BL/6-Ly5.1 background for use as recipient animals for all transplant and toxicity experiments. Single cell suspensions of 1 × 105 Eμ-Myc lymphoma cells in PBS were injected into 8-12 week old huMCL-1;Ly5.1 recipient mice by intravenous (i.v.) tail vein injection. Recipient mice were sex matched to transplanted tumors. Working solutions of cyclophosphamide (CP) and 5-FU (Sigma) were prepared in PBS. CP was administered by intraperitoneal (i.p) injection, 5-FU by i.v. tail vein injection. S63845 (Servier) was formulated extemporaneously and protected from light in 2% Vitamin E/TPGS (Sigma) in NaCl 0.9% (w/v) and delivered by i.v. tail vein injection at indicated doses and schedule. Mice were monitored for sickness and killed when deemed unwell in order to generate survival curves by experienced mouse technicians (blinded to the treatments and genotypes of the mice).
Statistical analysis
Prism software (GraphPad) was used to generate survival curves and perform all statistical testing of data. All data is presented as mean±s.e.m unless otherwise stated. P values > 0.05 were considered significant.
ACKNOWLEDGMENTS
We thank the Herold and Strasser labs, Andrew W. Roberts and David C. S. Huang for advice in the preparation of the manuscript, Giovanni Siciliano, Krystal Hughes, Dan Fayle, Hannah Johnson and Cassandra D’Alessandro for technical assistance for in vivo experiments and animal husbandry. Our work is supported by the Australian National Health and Medical Research Council (Project Grant 1145728 to MJH 1143105 to MJH and AS, 1086291 to GLK; Program Grant 1016701 to AS and Fellowship 1020363 to AS), the Leukemia and Lymphoma Society of America (LLS SCOR 7001-13 to AS and MJH), the Cancer Council of Victoria (1086157 and 1147328 to GLK, 1052309 to AS and Venture Grant MJH and AS), a VCA fellowship (MCRF17028 to GLK), a Leukaemia Foundation Grant in Aid (to AS and GLK) and a Postgraduate Award (to MSB), sponsored research funding from Servier, a bequest from the Estate of Antony Redstone (to AS and GLK) as well as by operational infrastructure grants through the Australian Government Independent Research Institute Infrastructure Support Scheme (9000220) and the Victorian State Government Operational Infrastructure Support Program.
Footnotes
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