Tschumper, Renee C.

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

AbstractIdentifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

Abstract E1912 was a randomized phase 3 trial comparing indefinite ibrutinib plus 6 cycles of rituximab (IR) to 6 cycles of fludarabine, cyclophosphamide, and rituximab (FCR) in untreated younger patients with CLL. We describe measurable residual disease (MRD) levels in E1912 over time and correlate them with clinical outcome. Undetectable MRD rates (<1 CLL cell per 104 leukocytes) were 29.1%, 30.3%, 23.4%, and 8.6% at 3, 12, 24, and 36 months for FCR, and significantly lower at 7.9%, 4.2%, and 3.7% at 12, 24, and 36 months for IR, respectively. Undetectable MRD at 3, 12, 24, and 36 months was associated with longer progression-free survival (PFS) in the FCR arm, with hazard ratios (MRD detectable/MRD undetectable) of 4.29 (95% confidence interval [CI], 1.89-9.71), 3.91 (95% CI, 1.39-11.03), 14.12 (95% CI, 1.78-111.73), and not estimable (no events among those with undetectable MRD), respectively. In the IR arm, patients with detectable MRD did not have significantly worse PFS compared with those in whom MRD was undetectable; however, PFS was longer in those with MRD levels <10−1 than in those with MRD levels above this threshold. Our observations provide additional support for the use of MRD as a surrogate end point for PFS in patients receiving FCR. In patients on indefinite ibrutinib-based therapy, PFS did not differ significantly by undetectable MRD status, whereas those with MRD <10−1 tended to have longer PFS, although continuation of ibrutinib would very likely be necessary to maintain treatment efficacy.

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Abstract Background: Multiple mechanisms exist for the positive selection of genes involved in oncogenesis such as gene amplification and transcriptional deregulation associated with chromosome translocations. Here we describe the case of a myeloma cell line that over time acquired extra copies of a derivative chromosome 4 involved in the t(4;14). These changes occurred over time both in vitro (in the generated JMW myeloma cell lines) as well as in vivo from extramedullary myeloma samples collected from the patient at various time points. In the case of the t(4;14)(p16;q32) 30% can be unbalanced translocations, always with loss of the der(14) and consequent loss of the FGFR3 gene expression, suggesting an indispensable role for MMSET located in the der(4). Results: We performed serial analysis on patient samples obtained at 9, 10 and 12 mos from diagnosis, along with a time course analysis of the cell line. At diagnosis the patient tumor cells displayed a complex clinical karyotype subsequently characterized by hypodiploidy, a t(4;14)(p16;q32), 13 monosomy and 17p13.1-. The remaining chromosome 13 and 17 were involved in an unbalanced reciprocal translocation. Notably, over time the cell line duplicated the total chromosome number becoming near-tetraploid. At the time of diagnosis, FISH analysis was performed on the patient sample revealing the translocation t(4;14)(p16.3:q32) in nearly all cells. The t(4;14) was also present in the stable cell line. It was noted that the patient cells exhibited more than one fusion signal per cell for the t(4;14) by FISH. As the disease progresses both in vitro and in vivo, the number of t(4;14)(p16.3:q32) fusion signals detected per cell increased with time suggesting a positive selection of the derivative chromosomes of the translocation. We further analyzed the t(4;14) in the JMW cells by FISH using MMSET/VH, and FGFR3/CH probe combinations. The full-length (MMSET/FGFR3+VH/CH) probe was used to detect the total number of fusions in the JMW cells. At the time of analysis (cell line passage #23), the majority of the cells (69%) had 6 fusion signals per cell while 23% had 5 fusions and 8% of the cells had 4 fusions. We found that most of the cells (88%) displayed 4 fusion signals detected using the MMSET/VH probes. In parallel studies using the FGFR3/CH probes, 61% of the cells had 2 fusion signals and 39% with 1 fusion signal. It is evident that the tumor cells display positive selection of the derivative chromosome der(4) of the t(4;14)(p16.3;q32), accompanied by an increased dosage of the target gene (MMSET) upregulated by the translocation. Conclusion: We present evidence for the amplification of the translocated chromosome in multiple myeloma, which we have termed “trans-amplicons”. Trans-amplicons may provide a selective growth and survival advantage to the myeloma tumor cells in an analogous manner to amplicons of DNA segments, but with much more powerful transcriptional consequences. Importantly, the evolution of genomic instability exemplified by the “trans-amplicons” in the patient cells was paralleled in vitro in the JMW cells.

Abstract One of the major mechanisms by which the immune system combats unpredictable foreign pathogens is to produce highly specific and effective antibodies by B cells. Somatic hypermutation (SHM) and class switch recombination (CSR) are two key events required for the production of highly specific and effective antibodies. Both SHM and CSR occur in the germinal center (GC), and are mediated by a common enzyme, activation-induced cytidine deaminase (AID) that specifically mutates the variable regions (IgV) and switch regions (IgS) of immunoglobulin genes by converting cytidines to uridines. It has been shown that in addition to the central deaminase domain, an N-terminal domain is essential to target SHM to the IgV region whereas a C-terminal domain is necessary for Ig CSR. Given its mutagenic nature, it has been postulated that AID may play causative roles in B cell lymphomagenesis. Consistent with this notion, AID is expressed and alternatively spliced in B cell tumors such as B-cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin’s lymphoma (NHL). However, it remains unclear whether post-GC malignant B cell AID expression and alternative splicing is a consequence of B cell transformation and/or is required to maintain growth and/or malignant B-cell survival, or whether post-GC AID expression and alternative splicing may also occur in normal B cells. To discriminate between these two possibilities, we analyzed AID expression and splicing in normal human B cells. Our data demonstrate that human tonsillar B cells prominently express the full-length AID transcript, and of interest, also express all four alternative transcripts that have been previously described in B-CLL and NHL B cells. To determine if all normal B cells express all of the AID transcripts, we performed limiting dilution of tonsillar B cells and used RT-PCR to detect AID expression. We surprisingly discovered that AID splicing variants are singly expressed by individual B-cells. To address the functional significance of each splicing variant, we considered the possibility that B cells expressing the alternative transcripts may be by-products of the normal GC reaction and are eliminated prior to GC exit. Conversely those B cells may contribute to the memory B cell pool if those AID variants are functional. Therefore, we examined AID expression and splicing in memory B cells using a more sensitive nested PCR approach. Our data show that AID is indeed expressed in peripheral blood memory cells but not in naïve B cells. Moreover, at least one splicing variant was expressed at levels comparable to the full-length AID transcript. Using limiting dilution, we again found that the full-length and alternatively spliced AID transcripts are singly expressed by individual memory B cells. Our results suggest that AID expression beyond the GC stage may play a role in sustaining selection pressure. Since the AID domains specific for SHM and CSR are encoded by different exons, we hypothesize that those variants may function differentially in SHM and CSR and this concept is under current study. Since each B cell only expressed one AID transcript, our data strongly suggest that SHM and CSR are coordinated in a stepwise process by generation of alternative AID splice variants that exhibit selective activity thereby allowing fine-tuning of humoral immunity.

Abstract The prognostic power of immunoglobulin (Ig) somatic mutation status in B cell chronic lymphocytic leukemia (B-CLL) is widely appreciated and has served as the rationale to systematically determine this molecular feature in B-CLL patients. Consistent with other reports, our study of Ig mutation status in a large cohort of B-CLL patients (n=599) found the most commonly used Ig heavy chain variable (VH) region gene was VH 1–69. The VH1–69 gene is of particular interest as there are numerous reports demonstrating that VH1–69 expressing B-CLL cells predominantly express the 51p1-like allele in an unmutated (UM) form and it is frequently associated with use of the D gene segment, D3-3; the J gene segment, JH6; and a relatively long third, complementarity-determining region (CDR3). Despite molecular definition of a commonly used form of VH1–69 in B-CLL, the clinical significance of CLL B cell usage of this prototypic form of VH1–69 remains to be determined. The goal of this study, therefore, was to perform a molecular and clinical analysis on our cohort of B-CLL patients expressing the VH1–69 gene. Our cohort consisted of 19 females and 55 males, with 89.5% expressing UM Ig VH. The 51p1-like allele was present in 75.7% of patients and the predominant D region used was D3 (64.7%). Patients in our cohort were classified by Rai risk as follows: 20 low (28.2%), 40 intermediate (56.3%), and 11 high (15.5%). For those patients with Rai stage available, analysis of specific D and J regions resulted in 16 D3-3/JH6 (26.7%); 14 D3-3/non-JH6 (23.3%); 16 non-D3-3/JH6 (26.7%); and 14 non-D3-3/non-JH6 (23.3%). Despite observing an overwhelming majority of leukemic cells with UM Ig VH, and the known relationship between mutation status and disease progression, it is interesting that our cohort included almost 30% with a low Rai risk. Of note, Ig VH mutation status did not segregate with Rai risk group or VH1–69 allele use (although few patients overall were mutated). However, an UM Ig VH status was associated with a CDR3 length >20 (p = 0.001) and with use of the JH6 segment (p = 0.003). When mutation status was analyzed in concert with specific use of the D3-3 allele, no relationship emerged; however, when this D region allele was analyzed for JH6 vs non-JH6 usage, combination of the D3-3 allele with JH6 was significantly associated with an UM Ig VH status (p = 0.004). Moreover, when J and D segment usage was analyzed in concert with Rai stage group, a significant (p = 0.019) relationship emerged between paired use of D3-3 and a JH6 segment and advanced (intermediate/high) Rai stage. Strikingly, only 1 of 18 VH1–69 patients with low Rai risk for whom D and J data were available expressed the specific combination of D3-3 and JH6. Although analysis of a larger cohort is clearly required to more formally and fully evaluate these relationships, to our knowledge, this is the first suggestion that D and J usage in VH1-69 expressing B-CLL patients correlates with advanced stages of disease. Because Ig VH usage is not believed to change over the course of disease, it is tempting to speculate that VH1–69 B-CLL patients that do not express the prototypic VH1–69 receptor may have a more favorable disease course. In summary, these data add further support to the notion that there is selection for specific Ig receptors in B-CLL and that the antigenic specificity of certain receptors may be associated with risk of progression.

Abstract Background: The inactivation of p53 by deletion of 17p13 is observed in 10% of multiple myeloma patients and is associated with shorter survival. In this study we were interested in assessing p53 function in the JMW myeloma cell line established from a patient with aggressive disease and circulating plasma cells. We characterized the plasma cells derived from the patient with the disease from the initial stage of procurement ofpatient tumor cells to permanence as a cell line. Results: At diagnosis, 95% of the cells had monosomy 17 and 4% of the cells had a FISH pattern consistent with interstitial deletion of p53 (17p13). However, we observed clonal evolution in that at 3 months from diagnosis, 70% of the patient cells exhibited a deletion of the p53 locus while 28% of the cells had a monosomy 17. This may be attributed to favored clonal expansion of the cells with the deletion of p53 (17p13). To resolve this issue we performed further interphase and metaphase FISH and found an unbalanced complex translocation resulting in LOH of 13q and 17p (der13 t(13;17)(q?;p?). In addition, we screened the remaining p53 gene for mutations by conformation-sensitive gel electrophoresis (CSGE). PCR fragments showing abnormal CSGE-banding patterns (in exons 5 and 8) were further characterized by DNA sequence analysis and we identified a G to A splice site mutation generating an alternate splice site 21 bp into exon 5. This mutation results in a mutant protein lacking amino acids 126–132 in the DNA binding domain of p53. We observed no differences in the expression level of p53 mRNA as determined by RT-PCR analysis. We determined the effect of the deletion on p53 protein expression and function. We examined the effect of DNA damage induced by UV radiation on p53 protein levels in JMW cells, using the myeloma cell line H929 (expressing wildtype p53) as a positive control for expression. The JMW cells were exposed to UV radiation (20 J/m2) and the cells were harvested for western blot analysis 5 hours after UV damage. An increase in the level of p53 protein was observed in JMW as well as the H929 cells following UV treatment. We then investigated the activation of p53 downstream targets. The CDK inhibitor p21WAF1/CIP1 is a well-characterized p53 target that mediates G1 cell cycle arrest. Thus, we examined the levels of p21 protein following UV treatment of JMW and H929 cells. Strikingly, JMW cells lacked p21 expression in untreated cells as well as UV exposed cells suggesting that the characterized deletion in the DNA binding domain is an inactivating mutation. Conclusion: Abrogation of p53 function was observed in the JMW cells. Further analysis of the p53 pathway in JMW and other myeloma cell lines will provide an in depth understanding of the role of p53 in disease progression. The study of the p53 pathway in myeloma is highly relevant, as we have shown it to be associated with relapsing and aggressive disease, particularly with extramedullary myeloma. Our studies suggest that malignant plasma cells have the ability to survive outside of the bone marrow microenvironment but a p53 dependent checkpoint results in cell death. In clones with p53 defects the cells survive and result in extramedullary disease, including plasma cell leukemia.

Abstract The IgVH mutation status is an independent predictor of time to treatment and survival in CLL. However, the biological basis of this difference is unknown. Clinical use of the IgVH mutation status as a prognostic marker is limited due to the technical difficulties of assessing this feature. As such, we probed for differences in protein expression by CLL cells with unmutated (UM) vs mutated (M) IgVH using phage peptide display libraries (PDPL). Methods: PDPL consists of phages engineered to express random peptides (7-mer) on the coat protein. From approximately 2x1011 phages displaying all possible permutations of 7-mer peptide, we selected phages binding to UM cells by incubation with 106 UM cells and elution of bound phages. Then, from numerous phages initially binding to UM cells, we subtracted phages binding to M cells by multiple incubations with M cells (106). Thus, the phages that bound only to UM cells were selected. The procedure was repeated multiple times to increase selection specificity. The peptide sequences displayed by selected phages were evaluated. We identified phage targets on UM cells by immunoprecipitation (IP) of CLL protein with specific phage and by sequencing of precipitated protein. The expression of target protein in CLL cells was confirmed by immunobloting and flow cytometry. Results: Sequencing of phages from the 3rd round of selection (UM v. M) revealed several peptides, with the most predominant being FPSAHFL. IP identified the cellular target for FPSAHFL as vimentin, an intermediate filament protein involved in cell motility and activation. The level of vimentin expressed by CLL cells varied between patient samples (n=15). UM CLL cases were found to express higher levels of vimentin (the mean MFI was 1002; SD 381), than cases of M CLL (mean MFI 547; SD 109), p=0.0128. To evaluate the prognostic significance of vimentin, we assessed vimentin expression by flow cytometry in a separate subgroup of 40 CLL patients. Archived cell samples collected between 6/2000 – 6/2001 were used. The median follow up was 81 months (40–233). Vimentin expression (MFI) as a continuous variable was a risk factor for a shortened time to treatment (TTT) with a proportional hazard ratio of 4.152 (95% CI 1.33–15.220, p=0.0317). Using MFI closest to the median (and rounded to 2 decimal places) as a cutoff, we stratified patients in high (n=21) and low (n=19) vimentin expression groups. The median times to treatment (TTT) in patients with high vs low level expression of vimentin were 2.8 and 12.9 years respectively, p=0.0025 (Fig.). Conclusion: Using novel proteomic technology, we found that CLL cells with unmutated IgVH expressed higher levels of vimentin than CLL cells with mutated IgVH. A high-level of vimentin expression defined by flow cytometry appears to be associated with high-risk disease. Validation of these findings could allow for the vimentin level to be used as a surrogate marker for expression of unmutated IgVH in CLL. Figure Figure