Citations

Abstract The generation of B lineage lymphocytes in bone marrow is contingent upon the combinatorial activities of transcription factors, signaling molecules, chromatin modifiers, and microRNAs. One transcription factor important for the generation of B cell precursors is the homeodomain protein HoxA9. Equally important as HoxA9 function in facilitating lymphoid lineage specification is silencing HoxA9 activity upon commitment to the B cell fate as sustained expression impairs later stage B cell differentiation. We previously showed that transcripts for the B cell specification factor EBF1 show an inverse expression pattern to HoxA9. Furthermore, EBF1 does not directly suppress HoxA9 transcription. Hox proteins are regulated by microRNAs in some developmental contexts. MicroRNA profiling of an EBF1-/- cell line stably expressing an inducible EBF1:ER fusion protein revealed a single miR induced by EBF1 activity, miR-204. Importantly, forced expression of miR-204 in EBF1-/- cells repressed transcription and protein levels of the HoxA9 co-factor, Meis1. We hypothesized that if Meis1 is a critical target of miR-204, then sustained expression of miR-204 should phenocopy conditional deletion of Meis1 or HoxA9. Indeed, we determined that forced expression of miR-204 in wildtype multipotent progenitors impaired B cell differentiation, similar to loss of HoxA9. These data suggest a novel role for EBF1 in B cell development, induction of miR-204 to silence a HoxA9-Meis1 transcriptional axis.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Evidence is accumulating that the molecular circuitry initiating lymphoid lineage specification correlates with expression of the receptor tyrosine kinase Flt3. Recombination activating gene-1 (RAG1) locus activation is a hallmark of lymphoid lineage specification. Previous studies established that expression of a RAG1-GFP/+ reporter begins in a subset of lineage negative (Lin-) cells that express high levels of c-kit and Sca-1 (LSK+). To investigate a regulatory connection between Flt3 signaling and RAG1 locus activation from this stage, RAG1-GFP/+ knockin mice expressing wildtype, heterozygote, and knockout Flt3-ligand (FL) alleles were generated. Mono-allelic expression of the FL gene reduced FL transcripts by half, providing an in vivo model to evaluate threshold requirements for FL in lymphocyte development. Percentages and numbers of LSK+ cells were similar in FL+/+ and FL+/- mice, but significantly decreased in FL-/- animals. Analysis of GFP expression within the LSK+ compartment revealed a FL dose dependent reduction in frequencies and numbers of GFP+ cells. The requirement for threshold levels of FL in regulation of GFP expression extended into the B220- c-kitlo IL-7R+ lymphoid progenitor and CD43+ B220+ Pro-B compartments, but not in later stage B cell precursors. These findings establish a critical requirement for FL in regulating one event in lymphoid specification, RAG1 locus activation. These studies were supported by startup funds to K.L.M. from Mayo Clinic.

The current paradigms of known peripheral immune abnormalities in B-Chronic Lymphocytic Leukemia (CLL) are not able to consistently explain patient complications and it is difficult to correct a given CLL patient's immune status. Here, we expand on our initial report demonstrating bone marrow (BM) hematopoietic dysfunction in untreated CLL patients (Manso et al., Leukemia volume 33, pages 638-652, 2019). CLL patient BM had significantly reduced frequencies and short-term functional capacity of hematopoietic stem and progenitor cells (HSPCs). Additionally, the remaining progenitors exhibited increased protein levels of the key hematopoietic transcriptional regulators GATA-2 and PU.1. We further evaluated the frequency and function of myeloid stem cells from controls and untreated CLL patients by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays. Over the 5 week duration of the assay, we observed delayed and partial recovery of myelopoiesis from CLL-derived HSPCs (Figure 1A). These data suggest that removal of HSPCs from the CLL leukemic microenvironment partially recovers their ability to sustain myelopoiesis. A known inflammatory mediator and hematopoiesis-modulating cytokine that is constitutively produced by CLL cells, TNF-α, induced increased expression of GATA-2 and PU.1 in specific HSPC subsets and reduced formation of short-term colony forming units in vitro. Addition of TNF-α to LTC-IC assays resulted in a striking ablation of myelopoiesis in a dose-dependent manner, partially reproducing the ex vivo results (Figure 1B). To further assess the direct impact of CLL cells on HSPC biology, isolated HSPCs from controls were exposed in vitro to leukemic CLL cells. The co-culture induced overexpression of GATA-2 and PU.1 in distinct HSPC populations, recapitulating our ex vivo findings (Figure 1C-D). When cell-cell contact was inhibited by use of Transwell inserts, an intermediate increase in GATA-2 and PU.1 was observed, highlighting the contributions of both soluble mediators and cell-cell contact to HSPC alterations. In both direct and Transwell co-culture conditions, overexpression of GATA-2 and PU.1 was reversed when TNF-α was neutralized (Figure 1E-F). Taken together, these findings indicate a significant role for CLL-derived TNF-α in HSPC modulation and expand our previous observations of BM dysfunction in untreated CLL patients. This data offers new molecular insight into the contribution of the leukemic microenvironment to altered hematopoiesis, contributing to immunodeficiency in CLL, and identifies TNF-α as a potential therapeutic target for correction of hematopoiesis in CLL disease. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Parikh:AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Novak:Celgene Coorperation: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board.

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Introduction: CLL is the most prevalent leukemia in the United States (≥120,000 cases) with approximately 5000 deaths per year. The dominant cause of mortality in CLL patients is serious infection. Indeed, about 80% of CLL patients will sustain infectious complications at some point during their disease course and infection risk increases with length of the disease. Steady-state hematopoiesis in BM is required to sustain a peripheral repertoire of blood cells and maintain immune competence. There have been few studies evaluating the hematopoietic capacity of CLL BM and how it may relate to immune competence. Thus, the goal of this study was to determine the status of BM hematopoiesis in untreated and treated CLL patients. Methods and findings: The hematopoietic stem (HSC) and progenitor cell pool (HSPC) in human BM is defined by expression of CD34 and lack of surface markers (Lin-) expressed on their mature progeny. Frequencies of Lin- CD34+ progenitors in BM between age matched healthy subjects (HS) (n=22) and untreated CLL patients (n=10) were compared. We note that the Lin+ cocktail used included an antibody to CD19, eliminating dilution of CD34+ HSPC frequency due to variation in leukemic B cell infiltration. CD34+ HSPCs were significantly reduced in untreated CLL patients (6.33±3.01% vs. 2.04±2.33% in HS vs. CLL, p=0.0004). Next we evaluated total CFU responding cells within the residual CD34+ cells to determine if they were functionally compromised. Numbers of total CFU-responsive CD34+ progenitors (CFU-GM, CFU-E, and CFU-GEMM) were reduced when assayed from purified CLL patient CD34+ cells compared to HS. Thus, while HSPC are present in CLL patient BM, they are functionally compromised even after removal from the tumor microenvironment. To determine if reductions in CFUs were accompanied by decreased myeloid and erythroid lineage differentiated progeny, we evaluated frequencies and absolute numbers of CD14+ monocytes and CD71+ erythroid progenitors by flow cytometry and found both were severely reduced in CLL patient BM. To determine if the reductions in myeloid/erythroid lineage cells was due to deficiencies in their precursor HSPCs, we evaluated frequencies and absolute numbers of HSC, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Proportions, but not absolute numbers, of HSC, MPP, and CMP were significantly reduced in CLL patient BM. Frequencies of CD14+ monocytes and CD71+ erythroid precursors were also significantly reduced in patients with low BM tumor burden (designated as 20-40% BM involvement), suggesting that the hematopoietic dysfunction is not solely due to tumor burden. Comparative analysis of frequencies of CD14+ monocytes in BM to frequencies of monocytes in peripheral blood revealed a strong association, suggesting that for some immune cell types the blood immune repertoire may be reflective of BM dysfunction. In addition to BM evaluation of untreated CLL patients, we also studied a cohort of post-therapy patients (n=23). Not surprisingly, we found considerable heterogeneity in the extent of BM hematopoietic reconstitution. Nevertheless, we observed robust BM hematopoiesis post therapy in a subset of treated patients (n=9 of 23). Summary: Our findings reveal BM hematopoietic dysfunction (HD) in CLL not solely due to the degree of tumor burden. Importantly, the BM HD is reversible by effective therapies. The HD is manifested by reduced numbers and function of HSPCs and production of their mature progeny. Of significance is the severe reduction in monocytic and granulocytic cells found in CLL BM. This is an important finding as these two innate cell types provide critical first responder protection against invading pathogens. Our study also uncovered that levels of blood monocytes can mirror their diminished production in BM. Future studies will focus on determining the mechanism(s) that reduce the ability of CLL BM to sustain immune cell production in addition to determining if restoration of hematopoiesis is a necessary outcome for more effective therapies. Disclosures Ding: Merck: Research Funding. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Pathological complete response (pCR) is accepted by FDA as a surrogate endpoint for accelerated approval of targeted agents in combination with chemotherapy based on better long-term outcomes compared to residual disease (Cortazar 2014). Methods: The multi-center, adaptively-randomized I-SPY2 platform trial uses pCR as the primary endpoint to identify investigational agents that will improve outcomes in women with stage 2/3 breast cancer with high risk of early recurrence, across all signatures, based on hormone receptor (HR), HER2, and 70-gene (MammaPrint) status. For patients with HR+ HER2- tumors, only 70-gene (Mammaprint) high-risk patients are enrolled. To date, 1200+ patients have been randomized to one of 14 arms: control (paclitaxel followed by AC); veliparib/carboplatin; neratinib; MK2206; trebananib; trastuzumab/pertuzumab; ado-trastuzumab emtansine/pertuzumab; pembrolizumabx4; ganitumab/metformin; ganetespib; PLX-3397. 7 agents graduated in at least one signature (> 85% probability of success in a 300-patient phase III confirmatory trial); 2 did not graduate; 1 stopped for toxicity, and 3 are enrolling (patritumab/trastuzumab, talazoparib/irinotecan, pembrolizumabx8). Local pathologists were centrally trained using the Residual Cancer Burden (RCB) assessment to ensure uniform evaluation and response classification; RCB 0 = pCR. Results: We evaluated the relationship between pCR and event free (EFS) and distant disease free survival (DDFS) in the first 522 pts (median follow-up:2.5 years). 180 pts achieved pCR (36%) while 338 did not (RCB=1-3). There were 82 EFS and 65 DRFS events. Over the entire group (including all arms), pCR was highly associated with 3-year EFS (p<0.001 for both). Pts achieving pCR had a 3% recurrence risk (RR) at 3 years; those with non-pCR had 24% RR over this time period. For distant recurrence, the 3-year RR with pCR was 2%, compared to 20% in pts with non-pCR. As expected, pCR rates varied by breast cancer subtype (HR+/HER2: 18% (35/196), HR+/HER2+: 40% (33/82), HR-/HER2+:68% (34/50), HR-/HER2-:41% (76/188)). The relationship between pCR and EFS was significant and clinically impactful within each subtype. 3-year survival (pCR group)Hazard Ratio OverallOverallHR+/HER2-HER2+TNBCEFS97%0.08 (0.03-0.23)0.14 (0.02-1.04)0 (NA)0.11 (0.03-0.37)DDFS98%0.08 (0.03-0.26)0.17 (0.01-1.23)0 (NA)0.09 (0.02-0.40) Conclusions: The first long-term efficacy results from the I-SPY2 TRIAL demonstrate that achieving pCR is a very strong surrogate endpoint for improved EFS and DDFS in a high-risk population, across all treatment arms, regardless of subtype. I-SPY2 shows substantially lower estimated EFS hazards for patients achieving pCR, compared to the 5 yr EFS hazard ratio for pCR vs not in Cortazar (hazard ratio 0.49), demonstrating important differences between a metaanalysis compared to a platform trial with uniform high-risk eligibility, standardized pathology assessment, and multiple targeted therapies. Our data support the use of pCR as a primary endpoint for accelerated approval of new drugs if EFS is evaluated in the same population. Based on these findings, the I-SPY2 TRIAL will test whether therapy can be deescalated or escalated for individual patients with the goal of achieving pCR for all. Citation Format: Yee D, DeMichele A, Isaacs C, Symmans F, Yau C, Albain KS, Hylton NM, Forero-Torres A, van't Veer LJ, Perlmutter J, Rugo HS, Melisko M, Chen Y-Y, Balassanian R, Krings G, Datnow B, Hasteh F, Tipps A, Weidner N, Zhang H, Tickman R, Thornton S, Ritter J, Amin K, Klein M, Chen B, Keeney G, Ocal T, Feldman M, Klipfel N, Sattar H, Mueller J, Gwin K, Baker G, Kallakury B, Zeck J, Duan X, Ersahin C, Gamez R, Troxell M, Mansoor A, Grasso LeBeau L, Sams S, Wisell J, Wei S, Harada S, Vinh T, Stamatakos MD, Tawfik O, Fan F, Adams A, Rendi M, Minton S, Magliocco A, Sahoo S, Fang Y, Hirst G, Singhrao R, Asare SM, Wallace AM, Chien AJ, Ellis ED, Han HS, Clark AS, Boughey JC, Elias AD, Nanda R, Korde L, Murthy R, Lang J, Northfelt D, Khan Q, Edmiston KK, Viscusi R, Haley B, Kemmer K, Zelnak A, Berry DA, Esserman LJ. Pathological complete response predicts event-free and distant disease-free survival in the I-SPY2 TRIAL [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS3-08.

Isothermal titration calorimetry (ITC) and circular dichroism (CD) were used to study the thermodynamics of RPC·G‐quadruplex DNA (G4) complex formation. The ruthenium polypyridyl complexes (RPCs) were [Ru(phen)3]2+ (12+), [Ru(phen)2(dppz)]2+ (22+), [Ru(phen)2(tatpp)]2+ (32+), and [Ru(phen)2(tatpp)(phen)2Ru]4+ (44+), and target DNAs were c‐MYC NHE‐III1 promoter sequence mutants forming 1‐2‐1 and 1‐6‐1 G‐quadruplexes. Formation of the 2:1 RPC·G4 complexes is characterized by entropy driven RPC binding to the top and bottom of G‐tetrad faces. 12+ appears to bind very weakly or not at all to G4 DNA. 22+ having a dipyridophenazine group to stack on the top and bottom of the G4 core, exhibits an average Ka = 6.7 × 104 m–1. 32+, with a larger G4 interactive tetraazatetrapyridopentacene group, binds with significantly higher affinity, Ka = 1.1 × 106 m–1. 22+ and 32+ appear to bind independently of G4 folding topology and RPC conformation. The thermograms for the titration of G4 DNA with rac‐44+ are characterized by two binding modes exhibiting higher and lower affinity (Ka,1 = 3.6 × 107 m–1 and Ka,2 = 3.2 × 105 m–1). The two binding modes are attributed to preferential binding of one of the 44+ enantiomers (e.g. ΛΛ) over the other isomers (e.g. ΔΔ or ΔΛ). Tighter binding of the preferred 44+ enantiomer, in comparison to 32+, is due to additional favorable entropy for locating a second [(phen)2Ru–]2+ moiety in a G4 groove. Weaker binding of the disfavored 44+ isomers must be due to a poorer fit of these isomers with the G4 faces.