Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity.

We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.

Pre-B acute lymphoblastic leukemia (ALL) is a common pediatric malignancy often successfully treated with chemotherapy [1]. Unfortunately, chemotherapy is not without side effects, including risk for secondary malignancies and other long-term complications [2]. Additionally, adolescents and adults fare less well, requiring greater reliance on allogeneic hematopoietic stem cell transplant [3]. While chimeric antigen receptor (CAR) T cell therapy for ALL [4] continues to advance, patients may benefit from additional therapeutic options.

As with other types of leukemia, pre-B ALL exhibits stage-specific hematopoietic developmental arrest, in this case, corresponding to hyperproliferation of immature B cell progenitors [5]. Treatment aimed at restoring differentiation capacity to leukemic cells has long been sought, but has proven elusive [6]. The only widely used form of differentiation therapy employs all-trans retinoic acid (ATRA), which has achieved remarkable success for the specific treatment of acute promyelocytic leukemia [7].

The transcription factor PAX5 plays a central role in the origin of pre-B ALL as the single most common somatically mutated gene observed in the disease [8–10]. About one-third of patients acquire heterozygous PAX5 mutations, with complete loss of both alleles rarely seen [9,11]. Deletions or other loss-of-function mutations are typical, but, less frequently, PAX5 rearranges to form fusion genes with ETV6 or other partners, generating dominant negative proteins [12]. Heterozygous germline PAX5 loss-of-function mutation is also a cause of inherited predisposition to ALL [13,14]. In ALL cases defined by wild type PAX5, some acquire mutations in EBF or E2A (TCF3) [9], both of which are upstream activators of PAX5 [5]. Functionally, PAX5 activates B lymphoid-specific gene expression while repressing genes specifying alternative lineages, including T lymphocyte-promoting, NOTCH1 [15]. As such, B lymphoid development in the bone marrow of Pax5-null mice arrests at the pre-B stage [16]. Pax5 loss-of-function in conjunction with Stat5 activation results in developmental blockage of the B cell transcriptional program and leukemic transformation in mice [17]. Importantly, forced re-expression of PAX5 in PAX5-deficient ALL was recently shown to normalize growth and differentiation of leukemic cells in culture and clear circulating leukemic cells in a Pax5-deficient/Stat5-activated mouse model of ALL [18,19]. While cooperating mutations in additional genes arise during leukemogenesis [20], these findings, taken together, indicate that reduced PAX5 activity reversibly drives the formation of pre-B ALL and represents an intriguing therapeutic target.

Nevertheless, modulating PAX5 activity is likely to prove challenging. Transcription factors are generally regarded as “undruggable” [21]. Gene replacement therapy or genome editing [22] may ultimately prove too inefficient when dealing with large numbers of malignant cells. Moreover, targeting or even defining ALL leukemic stem cells for correction may be problematic, if not impossible [23]. However, in the case of genes that are members of paralogous gene families, such as PAX5, genetic redundancy may offer a feasible alternative.

The mammalian PAX gene family consists of nine paralogs [24]. Divergence among its four subfamilies is largely non-coding, within cis regulatory regions, allowing for tissue specific expression among family members [25]. In particular, members of the PAX2/5/8 subfamily (Fig 1, S1 Fig) contain largely identical functional domains, share DNA binding specificity, and exhibit functional redundancy [26,27]. For example, mouse gene targeting experiments, in which PAX2 is replaced by PAX5 under control of endogenous PAX2 regulatory elements, show complementation of developmental abnormalities otherwise resulting from PAX2 deletion [28]. While there is spatiotemporal overlap of PAX2/5/8 expression, for instance in parts of the developing nervous system, less overlap occurs in adult tissues [29]. PAX8 is expressed predominantly in the adult thyroid and PAX2 in the adult kidney, where PAX2 plays a protective role in response to hyperosmolarity encountered by inner medullary cells of nephrons [30]. Only PAX5 is expressed in lymphocytes.

As neither PAX2 nor PAX8 are expressed in lymphocytes, they are unlikely to be subjected to the same selective pressures favoring PAX5 mutation during leukemogenesis, and, not surprisingly, mutations are not detected in ALL [9]. Therefore, it is not hard to imagine that PAX2 and PAX8 could represent intact yet latent functional substitutes for PAX5 in pre-B ALL. Here we demonstrate the ability of both PAX2 and PAX8 to substitute for PAX5 loss-of-function and reverse the developmental blockade in pre-B ALL cells. We show that restoration of differentiation is similar using all three PAX family members and consists of changes to downstream gene expression, cell surface marker expression, cell size, and ultimately cell growth and survival. Additionally, as the translational utility of this strategy is predicated on the ability to activate the endogenous expression of these paralogs in the B cell lineage, we evaluate the aforementioned pathway of response to hyperosmolarity, which plays a prominent role in the kidney. We show that PAX2 and PAX5 exhibit transcriptional upregulation in response to hyperosmolarity in pre-B ALL cells, that PAX2 activation in lymphocytes, as in the kidney, is mediated by the tonicity response enhancer binding protein (TonEBP/NFAT5), and, finally, that hyperosmolarity-driven PAX2/5 activation correlates with changes in B cell developmental gene expression similar to those seen with exogenous PAX2/5/8 re-expression.

PAX5 loss-of-function results in B cell developmental blockade and contributes to leukemic transformation [16,17]. As an important early B cell transcription factor, PAX5 is responsible for both positively and negatively regulating developmental genes, driving differentiation towards a B lymphoid specific fate. Transcriptional targets of PAX5 are numerous and include B cell receptor (BCR) complex protein CD79a, the B cell specific transcriptional regulator BACH2, and the canonical B cell specific surface antigen CD19.

We began by confirming recent findings that re-expression of exogenous PAX5 rescues PAX5-deficient pre-B ALL cells [18] and assessing whether exogenous expression of PAX5 paralogs, PAX2 or PAX8, could function in a similar capacity. We initially evaluated the ability of PAX5, PAX2 or PAX8 to regulate a subset of PAX5 transcriptional targets, including CD79a, BACH2, and CD19. We also included CD10, which is a marker of B cell differentiation exhibiting a bell-shaped pattern of developmental expression levels that peak at the pro to pre-B cell transition [18,31]. We tested PAX factors in Reh cells, which were derived from a primary clonal culture isolated from pre-B ALL peripheral blood [32] and contain a heterozygous p.A322fs PAX5 null mutation [33]. As a PAX5 wild type control, we compared 697 cells, which are derived from a primary clonal culture of ALL bone marrow [34] and contain an E2A(TCF3)/PBX1 fusion gene arising from a t(1;19) chromosomal translocation [35]. Cells were stably transduced with lentivirus expressing either full length human PAX5, PAX2, or PAX8, along with a fluorescent marker, ZsGreen, driven from an internal ribosomal entry site (IRES). As a functionally negative control, we used a vector expressing the clinically observed pre-B ALL PAX5 null mutation, PAX5p.V26fs [36]. At day 4 following transduction, 2×105 ZsGreen-positive cells of each transduction type were sorted by FACS (see S2 Fig for gating strategy). Using quantitative real time PCR, we found that transgene expression of PAX5, PAX2, or PAX8 in both Reh and 697 cells led to significant upregulation of PAX5 target gene expression, relative to empty vector or the negative control PAX5p.V26fs.

With the exception of CD10, which is not a known PAX5 transcriptional target, this upregulation was more pronounced in PAX5-mutant Reh cells compared to PAX5-wild type 697 cells (Fig 2A and 2B, respectively).

To further evaluate the ability of PAX2 and PAX8 to rescue PAX5 loss-of-function in pre-B ALL cells, we assessed whether their transcriptional redundancy resulted in enhanced immunophenotypic progression by comparing their ability to modulate a subset of surface markers of B cell differentiation. CD10 (CALLA) and CD19 are surface markers found on normal, as well as leukemic, pre-B cells. Increases in both markers are expect to accompany B cell differentiation, whereas CD38 and CD43 are both downregulated during the large-to-small pre-B cell transition [18,37]. Reh and 697 pre-B ALL cells were transduced with lentivirus expressing PAX-IRES-ZsGreen, as before. At day 4 post-transduction, cells were stained with antibodies for cell surface markers, followed by analysis of ZsGreen-positive cells using flow cytometry. Cells expressing PAX5, PAX2, or PAX8 constructs showed significantly upregulated levels of CD10 and CD19 with downregulated levels of CD38 and CD43, relative to cells transduced with either empty vector or PAX5p.V26fs (Fig 3A and 3B). These results demonstrate a level of functional phenotypic rescue beyond simple transcriptional activation and show a shared ability within the PAX2/5/8 subfamily to promote immunophenotypic changes associated with advanced differentiation in pre-B ALL. Interestingly, PAX5-wild type 697 cells again exhibited similar results (Fig 3C, note scale of intensity).

The large-to-small pre-B cell transition occurs just prior to the emergence of the immature B cell and marks the end of the heavily proliferative large pre-B cell state, resulting instead in a population of pre-B cells which are not only smaller, as the name suggests, but also less proliferative [38]. As noted, we observed that transduction of Reh and 697 cells with PAX paralogs led to decreases in expression of CD38 and CD43, which are both downregulated during this transition [18,37]. This observation suggested that, consistent with prior observations related to PAX5 re-expression in Reh cells [18], PAX2 and PAX8 could advance differentiation in these cells, driving them through the large-to-small transition and ultimately to a normal, more quiescent state. To address this possibility, we analyzed changes in cell size as well as effects on replicative potential following transduction with PAX factors.

The flow cytometry parameter of forward scatter area (FSC-A) is a widely accepted proxy for estimating cell size [39]. Similar to PAX5, exogenous expression of PAX2 and PAX8 led to a reduction in Reh cell FSC-A ranging from 7–10.1%, relative to either empty vector or PAX5p.V26fs negative control (Fig 4A and 4B). Again, 697 cells displayed similar results (Fig 4B). However, as a negative control, the human embryonic kidney cell line, HEK293T, transduced with PAX2/5/8 or controls, did not exhibit a shift in cell size (S3C Fig).

We next evaluated the effect of exogenous PAX paralog expression on the long-term replicative potential of Reh and 697 cells. Cells were transduced with PAX5, PAX2, PAX8, empty vector, or PAX5p.V26fs negative control. At day 4 post-transduction, 2×105 cells of each group were FACS-sorted for ZsGreen (at ~98% purity, see S2 Fig for gating strategy) and returned to culture. For the following 6 days, daily measurement of culture density, performed in duplicate using a hemocytometer, allowed us to compile growth curves for all groups. While control cultures expanded normally, PAX paralog expression resulted in a complete inhibition of culture expansion in Reh cells (Fig 4C, S4A Fig). Growth inhibition was also present, but less complete, in 697 cells (Fig 4D, S4B Fig) and largely absent in HEK293T control cells (S3B Fig). From this point, it became necessary to periodically passage cultures in order to maintain viable cell densities (i.e., 2×105–2×106 cells/mL). At days 11–14, we again used flow cytometry to measure ZsGreen-expressing cell populations. Cultures transduced with PAX paralogs exhibited dramatically reduced ZsGreen expression as a percentage of total cells, ranging from 28–54% in Reh cells and 6–13% in 697 cells, whereas both the empty vector and PAX5p.V26fs control groups maintained expression in ~90% of cells (Fig 4E and S4C Fig). Growth inhibition and the reduced proportion of ZsGreen-positive cells together suggest that these cells reduce their rate of growth and are outgrown by the ~2% of ZsGreen negative cells initially harvested by mis-sorting and/or that PAX/ZsGreen-positive cells die out so that only ZsGreen-negative cells remain and continue to grow.

In support of the latter interpretation, PAX gene expression led to an apparent delay in cell cycle progression and conferred a modest increase in apoptosis, as measured by flow cytometry analysis of DNA content (with DAPI staining) and Annexin V staining, respectively (S5 Fig).

We have therefore confirmed previously published literature showing that restoration of PAX5 levels rescues deficiency of PAX5 activity in pre-B ALL cells [18] and have shown for the first time that its paralogs, PAX2 and PAX8, demonstrate a high level of functional redundancy in downstream activation of B cell specific gene expression, promoting differentiation similar to that seen with PAX5.

The observation that PAX2 and PAX8 can rescue the PAX5 loss-of-function differentiation blockade in pre-B ALL cells suggests their activation in vivo could represent a potential therapeutic strategy. In such a context, the use of small molecules to induce their endogenous expression would be useful.

In attempting to identify drugs capable of activating endogenous PAX2 or PAX8 we initially surveyed a variety of agents targeting epigenetic repressive marks or that have been reported to promote lymphocyte differentiation; however, none induced detectable PAX2 or PAX8 expression. We then evaluated compounds known to induce PAX family gene expression in other model systems. Manipulation of transmembrane voltage potential in Xenopus laevis activates transcription factors, including PAX6, resulting in ectopic eye formation [40]. Based on this observation, we tested a variety of hyper- and hypo-polarizing compounds for their ability to induce PAX2 and/or PAX8 expression in Reh cells. We found that 24 hour exposure to membrane depolarizing concentrations (80mM) of C6H11KO7 (K-gluconate) in cell media led to induction of PAX2 expression to as much as 0.3 fold of baseline PAX5, as measured by qRT-PCR. Interestingly, significant upregulation of PAX5 expression was also observed (Fig 5A and S6A, S6B and S6C Fig).

While such concentrations of K-gluconate are known to induce membrane depolarization [41], treatment with monensin and other compounds that are also known to promote membrane hypopolarization did not influence expression of PAX genes. As both potassium and gluconate ions are potentially capable of independent interaction with membrane channels or other cellular machinery that could influence downstream gene expression [42], we tested a variety of salts containing these and other ions, for their ability to influence PAX expression. Surprisingly, 80mM concentrations of NaC6H11O7 (Na-gluconate), KCl, CaCl2, and NaCl all promoted detectable induction of both PAX2 at 0.08–0.5 fold and PAX5 at 3.8–6.1 fold, relative to baseline PAX5 (Fig 5A). Evaluation of downstream PAX5 target and developmental marker genes, CD19, BACH2, and CD10, demonstrated concurrent upregulation at levels similar to those seen with transgene-driven exogenous PAX expression (Fig 5B). While the ionic composition of these agents differs, a commonality is that they all increase the osmolarity of cell growth media.

We observed quantitative differences in the ability of these osmolytes to induce PAX2/5, perhaps due to their variable ability to penetrate the cell membrane, utilizing channels specific for their uptake or efflux. As such, based on their greater relative ability to upregulate both PAX2 and PAX5 in Reh cells, we selected K-gluconate and CaCl2 for further evaluation. Dose-response curves revealed that 80-100mM concentrations (corresponding to ~400-540mOsmol/kg H2O in RPMI media) were optimal for either salts’ ability to upregulate PAX2 and PAX5, with little activity occurring at lower concentrations (Fig 5C and 5D, and S7A and S7B Fig). Similar results were seen with 697 cells; however, the magnitude of induction was less than that observed in Reh cells (S7C and S7D Fig).

In studying the kinetics of this response to hyperosmolarity, 24 hour exposure to high salt concentrations, followed by sorting of live cells and return to normal media for extended incubation revealed that both PAX2 and PAX5 upregulation occurred quickly, but decreased within 24 hours post exposure to salt (Fig 5E and 5F). While CD10 followed a similar temporal pattern to PAX gene modulation, increases in direct PAX5 target genes CD19 and BACH2 were delayed and more persistent, supportive of their sequential response to PAX induction following hyperosmolarity, rather than to hyperosmolarity alone (Fig 5G). Interestingly, the RNA collection method affected the magnitude of induction for PAX2, which was as much as 10-fold greater in RNA extracted from cells immediately following treatment compared to RNA harvested from cells which were first treated, then sorted for viability (as assessed by FSC-A/SSC-A). In contrast, induction of PAX5 appeared to be similar regardless of the RNA collection method. (RNA collection methods are described in Figure Legends and Methods.) This observation suggests an interplay between cell viability and PAX2 expression (Fig 5A and 5C, compared to Fig 5E; see also S2 Fig for gating strategy).

Using cell surface markers, morphological changes, and a subset of PAX5 transcriptional targets, we have demonstrated the ability of PAX2 and PAX8 to rescue PAX5 loss-of-function in pre-B ALL cell lines. To evaluate the full extent to which PAX2 and PAX8 can substitute for PAX5, as well as to compare PAX transgene expression with the response to hyperosmolarity, we evaluated global changes in gene expression by RNA sequencing (RNA-seq) following PAX2, PAX5, or PAX8 transfection or treatment with 80mM K-gluconate or CaCl2 in Reh cells.

Gene set enrichment analysis (GSEA) revealed common enrichment pathways based on biological process and transcription factor targets (Fig 6). Gene sets previously shown to be either direct transcriptional targets of PAX5 at the pro and mature stages of B cell development or whose regulation relies on PAX5 mediation of differentiation from the pro to mature B cell stages displayed enrichment as well [13,43]. We restricted analysis to gene sets with a false discovery rate less than 0.05.

We observed enrichment of 420 gene sets in Reh cells transfected with PAX5. 35% (149) or 26% (108) of these gene sets are also enriched in PAX2 or PAX8 transfected cells, respectively, with 14% (57) common to all three samples (Fig 6A). The majority of these gene sets involve genome accessibility and protein translation (e.g., methylation, peptidyl lysine modification, translational initiation, and cytoplasmic translation), but we also see negative enrichment of known cell cycle regulation transcription factor gene sets such as those involving MYC/MAX and E2F1 (MYCMAX_01 and E2F1_Q4, respectively, S1 Table). PAX2/5/8 transfected samples also show similar enrichment patterns in the PAX5 B cell developmental gene sets (Fig 7A and 7B, S2 Table and S1 Dataset), each factor promoting the upregulation of CD72, IRF4, BACH2, CD19, EGR1, IKZF3, KLF2, and SAMHD1 as well as the suppression of CYBB and FOS.

Interestingly, K-gluconate and CaCl2 share a larger percentage of the PAX5 enriched gene sets, 66% (278) and 60% (254), respectively, than either PAX2 or PAX8 transfected samples (Fig 6B). 53% (221) of the PAX5 enriched gene sets are also enriched following both CaCl2 and K-gluconate exposure (S1 Table). In general, there is a larger, overlapping response when comparing the two salt treatments, presumably part of a general response to hyperosmolarity. Of note, gene sets related to the transport of calcium ions, chloride ions, potassium ions, and organic anions, as well as cytosolic calcium regulation, are positively enriched for all three treatments—an expected result for cells exposed to K-gluconate and CaCl2, but not for forced expression of PAX5. Again, the MYCMAX_01 and E2F1_Q4 gene sets are negatively enriched, linking increasing osmolarity with a pathway for reduced proliferation and a decrease in B cell size [44], although leading edge analysis of the gene sets suggests different genes responsible for enrichment when compared to PAX2/5/8 (S3 Table). Both CaCl2 and K-gluconate show their strongest PAX5 related response in the pro to mature B cell transition gene set (Fig 7C and S2 Table). Here overlapping clusters of upregulated genes similar to the individual pro and mature B cell gene sets (e.g., KLF2, EGR1, IKZF3, SAMHD1, and CD72) are highlighted. TNFRSF13C/BAFF-R, a regulator of peripheral B cell survival, is also upregulated, whereas downregulated genes include cell cycle initiation factors CDC6 and CDC45 and pre-replication complex components MCM3, MCM6, MCM7, and MCM10.

In total, 43 of the 57 gene sets common to PAX2/5/8 transfected samples are also enriched in the CaCl2 and K-gluconate treated samples, corresponding to 10% of the total enriched gene sets in PAX5 transfected Reh cells (Fig 6C, S1 Table). Most of the enriched sets common for both PAX2/5/8 transfectants and salt treatment again relate to genome structure and protein synthesis and also similarly include MYCMAX_01 and E2F1_Q4 transcription factor targets. Both the pro-B cell and pro to mature B cell gene sets are enriched in all samples, as well. The greatest similarity across treatment conditions is seen in the pro-B cell set of genes (Fig 6D), with the only difference being a lack of negative enrichment of genes in either CaCl2 or K-gluconate treated cells. Overall, these results suggest that B cell maturation is regulated by a set of genes and pathways commonly responsive to either PAX gene expression or hyperosmolarity.

Liu et al. [13] restored PAX5 expression in Reh cells and compared global changes in gene expression via RNA-seq to gene expression in a Pax5-deficient/Stat5-activated mouse model of ALL. They identified 31 genes in Reh cells, upregulated by greater than two-fold in response to exogenous PAX5, that are also commonly upregulated with restoration of Pax5 in the mouse model of ALL. Restoration of Pax5 in this model triggers durable disease remission. The log2 fold change values we observed for these 31 genes in PAX2/5/8 transfected and CaCl2 or K-gluconate treated cells appear in Fig 6E, charted alongside corresponding original data from Liu et al. Although treatment windows for our samples were somewhat brief compared to duration of Pax5 induction in mice, we found similar increases in relative expression across this set of 31 genes, albeit at levels roughly half of what Liu et al. reported. These data demonstrate that PAX paralog expression or hyperosmolar treatment both similarly modulate an important subset of genes associated with disease remission when PAX5 expression is restored to normal levels in cell and mouse models of PAX5-deficient ALL.

To confirm RNA-seq results, we used qRT-PCR to validate the responses of several genes where the heatmap clustering showed them to be upregulated by at least 4 of 5 treatment conditions, along with an additional gene, SNX12, which was slightly downregulated by 4 of 5 conditions. qRT-PCR analysis of all 7 of these genes accurately corroborated the trends seen in the RNA-seq data (S8A Fig). Notably, relative to RNA-seq, magnitudes of induction (if present) were almost always greater using qRT-PCR ΔΔCT values. This is likely due to the conservative estimates of differential expression from the DESeq2 normalization algorithm we employed to analyze RNA-seq data. Nevertheless, trends were consistent regardless of technique or genes referenced for comparison.

Cellular response to hypertonicity, as brought about by hyperosmolarity, is thought to be largely mediated by the tonicity-responsive enhancer binding protein, TonEBP [45]. TonEBP, also called (and referred to here as) NFAT5 (nuclear factor of activated T cells 5), is a transcription factor predominantly associated with the kidney but which is also expressed in other tissues, including B cells and, as its name suggests, T cells. Initial response to hypertonicity by NFAT5 involves post-translational modification via phosphorylation, followed by transcriptional activity, including self-induction. Interestingly, NFAT5 mediated gene regulation in the high salt environment of nephrons has been shown to include elevated PAX2 expression, seemingly as part of a survival mechanism during osmotic stress [30]. Not surprisingly, our RNA-seq data showed that hyperosmolarity in Reh cells led to induction of NFAT5, as well as several of its downstream targets (S1 Dataset), consistent with the notion that hyperosmolar concentrations of K-gluconate and CaCl2 generate a canonical response to hypertonicity (i.e., an increase in osmotic pressure gradient across the cell membrane). Subsequent evaluation by qRT-PCR confirmed that NFAT5 mRNA levels, as well as a downstream target associated with B cell maturation, B cell activating factor (BAFF), along with its receptor, TNFRSF13C (BAFF-R) [46], were upregulated in Reh cells after 24 hour treatment with 80mM K-gluconate or CaCl2 (Fig 8A). BAFF-R alone was also upregulated by PAX transgene expression.