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Coculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-?B within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-?B target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-?B. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-?B and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-?B, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-?B and STAT3 in patients with CLL.

Acute lymphoblastic leukemia (ALL), one of the most common malignancies, has been reported to account for nearly one-third of all pediatric cancers. Failure of induction chemotherapy is the leading cause of relapse and mortality in pediatric patients treated for ALL. Monitoring the therapeutic efficacy of induction chemotherapy and improving its success rate are critical to the clinical management of ALL. FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed on progenitor cells and ALL blasts. FLT3 ligand (FLT3L) is detectable during homeostasis and increases in hypoplasia due to genetic defects or treatment with cytoreductive agents. Here, we continuously monitored changes in FLT3L levels during induction chemotherapy in children with ALL to provide a more sensitive assessment of MRD, similar to NGS. The study included 54 children with ALL whose peripheral blood FLT3L levels were measured by ELISA weekly during induction chemotherapy. Weekly sampling revealed a significant difference in the kinetics of FLT3L response between children in complete remission (CR) and partial remission (PR), at day 14 of treatment (CR:519±10pg/ml vs PR:54±10pg/ml, P＜0.001). Furthermore, the achievement of CR in these patients was associated with higher FLT3L (150±10pg/ml vs 32±10pg/ml, P＜0.05) at day 28. Clinical prediction models show that combining minimal residual disease (MRD) status with FLT3L measurements better predicts whether patients will achieve CR after induction chemotherapy. Moreover, FLT3L levels were positively correlated with DC and T cells and negatively correlated with WBC, PLT and LDH during induction chemotherapy in children with ALL. Therefore, we hypothesized that higher levels of FLT3L might improve antitumor immunity by activating DC and T cells. However, further research is needed. In conclusion, measurement of FLT3L in children with ALL during induction may provide added value in assessing early treatment response to chemotherapy, the impact on long term outcome needs further study.

Angiogenesis is critical to tumor progression. The homeobox gene GAXinhibits angiogenesis in vascular endothelial cells (ECs). We have identified a microRNA (miR-130a) that regulates GAXexpression and hypothesized that it plays a major role in modulating GAXactivity in ECs. A 280-bp fragment from the GAX3′-untranslated region (3′-UTR) containing 2 miR-130a targeting sites was observed to be required for the rapid down-regulation of GAXexpression by serum and proangiogenic factors, whereas the activity of the GAX promoter did not vary with exposure to serum or proangiogenic factors. This same 280-bp sequence in the GAX3′-UTR cloned into the psiCHECK2-Luciferase vector mediated serum-induced down-regulation of the reporter gene when placed 3′ of it. Finally, forced expression of miR-130a inhibits GAXexpression through this specific GAX3′-UTR sequence. A genome-wide search for other possible miR-130a binding sites revealed an miR-130a targeting site in the 3′-UTR of the antiangiogenic homeobox gene HOXA5, the expression and antiangiogenic activity of which are also inhibited by miR-130a. From these data, we conclude that miR-130a is a regulator of the angiogenic phenotype of vascular ECs largely through its ability to modulate the expression of GAXand HOXA5.

To investigate the role of BCL6in the pathogenesis of gastric lymphoma, we analyzed the BCL6promoter region for BCL6translocations, somatic hypermutations, and deregulating mutations in 43 gastric lymphomas, including 4 extranodal marginal-zone B-cell lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas), 33 diffuse large B-cell lymphomas (DLBCLs), and 6 composite DLBCLs with residual MALT lymphoma (DLCLMLs). BCL6promoter substitutions by immunoglobulin (Ig) and non-Igtranslocation partners, resulting in its deregulation, were frequently involved in DLBCL (36.4%) and DLCLML (50%). Two novel BCL6translocation partner genes, 28S rRNAand DMRT1, and a new BCL6translocation breakpoint in intron 2 were also identified. Deregulating mutations were found only in DLBCL (24.2%), which correlated significantly with high BCL6 protein expression. Significantly, high BCL6 expression correlated strongly with longer overall survival (OS), independent of mechanism in gastric DLBCL and DLCLML. Gastric DLBCLs were further subclassified into germinal center B-cell–like (GCB) and non-GCB subgroups immunohistochemically. High BCL6 expression was detected in all GCB cases, irrespective of BCL6genetic alterations. In the non-GCB subgroup, BCL6-deregulating mutations correlated significantly with high BCL6 expression level. No significant correlation was found between the BCL6 expression level and OS in the non-GCB subgroup, which had significantly poorer prognosis than the GCB subgroup.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic cell surface protein, which is expressed on the neoplastic B cells of patients with chronic lymphocytic leukemia (CLL), but not on virtually all normal post-partem tissues. ROR1 functions as a receptor for Wnt5a, which induces activation of Rho-GTPases, AKT, and HS-1 and promotes migration, proliferation, and survival of CLL B cells. We observed that freshly isolated CLL cells also had high levels of pSTAT3 (Y705), which attenuated over time in culture in serum-free media unless the CLL cells were stimulated with exogenous Wnt5a. This effect of Wnt5a could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb that currently is undergoing clinical evaluation in patients with CLL. We examined the capacity of recombinant Wnt5a (rWnt5a) to induce activation of STAT3 in isolated CLL cells by performing a time-course study, evaluating for pSTAT3 in serum-starved CLL cells at time 0, 5', 15', 30' and 1, 2, 3, 4, and 24 hours after stimulation with exogenous Wnt5a. Surprisingly, activation of STAT3 in CLL cells was delayed, appearing first at 3 hours after addition of rWnt5a, and peaking at 24 hours, the last time point examined. In contrast, activation of CLL cells via treatment of CLL cells with interleukin (IL)-6, induced activation of STAT3 within 15' of the addition of this cytokine to the cultures. We hypothesized that the activation of STAT3 in CLL cells by Wnt5a was indirect, being caused by a factor(s) made by isolated CLL cells in response to stimulation with Wnt5a. To test this hypothesis, we examined CLL cells, and harvested supernatants of cultured CLL cells, at various times after stimulation with rWnt5a. RNA isolated from CLL cells was examined via real-time PCR for cytokine transcripts. This revealed a prominent, time-dependent increase in transcripts encoding IL-6 following stimulation of CLL cells with rWnt5a. Consistent with this observation we found Wnt5a induced CLL cells to secrete IL-6, which was detected via ELISA at increasing concentrations over time in culture-supernatants of CLL cells treated with rWnt5a, but not in the culture supernatants of unstimulated CLL cells. Culture supernatants collected from CLL cells stimulated for 24 hours with rWnt5a could induce activation of STAT3 within 15' in serum-starved CLL cells; this effect could be inhibited significantly by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of concomitantly-added Wnt5a to induce latent STAT3 activation at 24 hours, suggesting that cirmtuzumab could inhibit Wnt5a-induced CLL-cell production of cytokines, such as IL-6. We examined for IL-6 in cultures of CLL cells treated for 24 hours with rWnt5a and cirmtuzumab, or rWnt5a and an antibody of irrelevant specificity. We found the concentration of IL-6 in the supernatants of CLL cells treated with rWnt5a and cirmtuzumab (110.4 ± 18.9 pg/ml) was significantly lower than that found in the supernatants of CLL cells treated with rWnt5a and control antibody (416.2 ± 53.7 pg/ml, p = 0.0007, Students t test). This study reveals that Wnt5a can induce CLL to secrete cytokines, such as IL-6, via a ROR1-dependent pathway, leading to latent, autocrine activation of STAT3.

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, particularly in patients with MCL. This suggests that ancillary pathways contribute to the growth/survival of MCL/CLL that are independent of BCR-signaling and/or inhibition of BTK. Studies have found that Wnt5a can promote activation of Rho GTPase Rac1 to enhance CLL-cell proliferation and survival via a ROR1-dependent,but BTK-independent pathway (Yu J, et al., Leukemia31:1333, 2017). Furthermore, Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which significantly could enhance the capacity of ibrutinib to clear CLL cells in vivo. MCL cells also express ROR1. Moreover, we find that the median level of ROR1 on primary MCL cells is higher than that on CLL cells; the median absolute mean fluorescence intensity of ROR1 (ROR1 DMFI) on MCL (87.5 ± 18.5 (S.D.), N = 8) was significantly higher than the median ROR1 DMFI on CLL cells (38.5 ± 18.5 (S.D.), N = 1567; (Cui B, et al., Blood128:2931, 2016)). We also found that the plasma of patients with MCL had high levels of Wnt5a that were comparable to those found in patients with CLL; in contrast Wnt5a was virtually undetectable in the plasma of age-matched healthy adults. We cultured primary MCL cells of different patients with ibrutinib, cirmtuzumab, or both ibrutinib and cirmtuzumab for 2 h, and then stimulated the cells with exogenous Wnt5a for 30 min. In parallel, the same MCL cells were cultured without exogenous Wnt5a. We found that Wnt5a induced Rac1 activation in the primary MCL cells. Cirmtuzumab, but not ibrutinib, could inhibit the capacity of Wnt5a to induce primary MCL cells to activate Rac1. We induced proliferation of MCL cells by co-culturing MCL cells with HeLa cells expressing CD154 (HeLaCD154) and recombinant interleukin (IL)-4 and IL-10. Addition of exogenous Wnt5a to co-cultures of MCL cells with HeLaCD154 cells and IL-4/10 significantly enhanced the proportion of dividing MCL entering S/G2 and the numbers of cell divisions over time. Treatment of the MCL cells with cirmtuzumab, but not ibrutinib, blocked Wnt5a-enhanced survival and proliferation of MCL cells from each of 3 different patients. This study demonstrates that Wnt5a can induce Rac1 activation, which leads to enhanced proliferation and survival of primary MCL cells via a ROR1-dependent signaling pathway that can be blocked by cirmtuzumab. On the other hand, this signaling pathway cannot be blocked by ibrutinib, even at concentrations that completely inhibit BTK or BCR-signaling. This implies that cirmtuzumab and ibrutinib may have additive, if not synergistic, activity in the treatment of patients with MCL.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen expressed on chronic lymphocytic leukemia (CLL) cells, but not on normal postpartum tissues. The ligand that binds to ROR1 is Wnt5a, which we find present at high levels in the plasma of patients with CLL relative to that of age-matched healthy adults. Wnt5a can induce CLL-cell activation of Rho-GTPases in a ROR1-dependent manner, thereby enhancing leukemia-cell proliferation, migration, and survival. Currently, the cell-source(s) of Wnt5a in CLL is not known. Good candidates include Nurse-Like cells (NLCs), which are monocyte-derived accessory cells that develop in the context of CLL cells and that are found within the leukemia-cell microenvironment in lymphoid tissues. Already NLCs have been shown to elaborate factors that can promote leukemia-cell activation/survival, including chemokines (e.g. CXCL12, CXCL13) and members of the tumor-necrosis-factor family (e.g. BAFF and APRIL). Because activated monocytes can produce Wnt5a, we hypothesize that NLC also may produce this non-canonical Wnt factor. For this study, we generated NLCs from blood mononuclear cells of CLL patients through co-culture with leukemia B cells using established methods. NLCs, which stained positive for CD163 and CD68, were isolated free of leukemia cells via adherence. We isolated RNA from purified CLL cells or purified NLC of the same patient for real-time PCR to measure the levels of WNT5Atranscripts. WNT5Atranscripts were abundant in the RNA isolated from NLC, but were negligible in RNA isolated from CLL cells each patient tested (N = 5). We evaluated for Wnt5a via ELISA in the culture-medium of isolated NLCs of five different patients. The cultured medium of NLCs from each patient had detectable Wnt5a that increased in concentration over time. Furthermore, ultra-high resolution confocal fluorescent microscopy of NLC-CLL co-cultures showed Wnt5a was expressed at high-levels in NLCs, but was undetectable in CLL cells of each patient tested (N = 3). We performed co-culture studies and trans-well assays to assess the survival and migration of CLL cells when cultured with or without NLCs, and with or without a neutralizing mAb specific for Wnt5a or cirmtuzumab, which is a humanized anti-ROR1 mAb that blocks Wnt5a-induced, ROR1-dependent signaling. We found that anti-Wnt5a or cirmtuzumab, but not a mAb of irrelevant specificity, each could comparably and significantly inhibit the protective effects of NLCs for CLL cells in vitro(p= 0.008, N = 3 and p= 0.0032, N = 3, respectively by paired student's t-test). Moreover, anti-Wnt5a or cirmtuzumab each could comparably and significantly inhibit the migration of CLL cells enhanced by co-culture with NLCs in vitro(p< 0.0001, N = 3, by paired student's t-test). We also examined for Rho-GTPase activation in CLL cells co-cultured with NLCs. CLL cells co-cultured with NLCs, but not CLL cells cultured alone, had high-level activation of Rac1 and RhoA. The activation of RhoA and Rac1 in co-cultured CLL cells could be blocked by neutralizing anti-Wnt5a or cirmtuzumab, but not by control mAb of irrelevant specificity. We conclude that NLCs express Wnt5a, which can enhance CLL-cell migration and survival via a ROR1-dependent pathway. We speculate that the high-level Wnt5a noted in the plasma of patients with CLL is produced primarily by NLCs, which reside in lymphoid tissues. If so, then the high-level Wnt5a present in the plasma of CLL patients may extend the protective effects of NLCs beyond the CLL microenvironment in which NLCs reside. Treatment with cirmtuzumab to block ROR1-dependent Wnt5a-signaling may mitigate such effects, potentially enhancing the clearance of leukemia cells when used alone or in combination with drugs that target other survival-signaling pathways in CLL.